Hydrazinyl-Indole Compounds and Methods for Producing a Conjugate

ABSTRACT

The present disclosure provides conjugate structures (e.g., polypeptide conjugates) and hydrazinyl-indole compounds used to produce these conjugates. The disclosure also provides methods of production of such conjugates, as well as methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/113,245, filed Dec. 7, 2020, now U.S. Pat. No. 11,426,465, which is adivisional of U.S. application Ser. No. 16/392,162, filed Apr. 23, 2019,now U.S. Pat. No. 10,888,623, which is a continuation of U.S.application Ser. No. 15/799,846, filed Oct. 31, 2017, now U.S. Pat. No.10,314,919, which is a divisional of U.S. application Ser. No.15/057,847, filed Mar. 1, 2016, now U.S. Pat. No. 9,833,515, which is adivisional of U.S. application Ser. No. 13/794,159, filed Mar. 11, 2013,now U.S. Pat. No. 9,310,374, which claims priority pursuant to 35 U.S.C.§ 119(e) to U.S. Provisional Application No. 61/727,603, filed Nov. 16,2012, the disclosures of each of which are incorporated herein byreference.

INTRODUCTION

The field of protein-small molecule therapeutic conjugates has advancedgreatly, providing a number of clinically beneficial drugs with thepromise of providing more in the years to come. Protein-conjugatetherapeutics can provide several advantages, due to, for example,specificity, multiplicity of functions and relatively low off-targetactivity, resulting in fewer side effects. Chemical modification ofproteins may extend these advantages by rendering them more potent,stable, or multimodal.

A number of standard chemical transformations are commonly used tocreate and manipulate post-translational modifications on proteins.There are a number of methods where one is able to modify the sidechains of certain amino acids selectively. For example, carboxylic acidside chains (aspartate and glutamate) may be targeted by initialactivation with a water-soluble carbodiimide reagent and subsequentreaction with an amine. Similarly, lysine can be targeted through theuse of activated esters or isothiocyanates, and cysteine thiols can betargeted with maleimides and α-halo-carbonyls.

One significant obstacle to the creation of a chemically altered proteintherapeutic or reagent is the production of the protein in abiologically active, homogenous form. Conjugation of a drug ordetectable label to a polypeptide can be difficult to control, resultingin a heterogeneous mixture of conjugates that differ in the number ofdrug molecules attached and in the position of chemical conjugation. Insome instances, it may be desirable to control the site of conjugationand/or the drug or detectable label conjugated to the polypeptide usingthe tools of synthetic organic chemistry to direct the precise andselective formation of chemical bonds on a polypeptide.

SUMMARY

The present disclosure provides conjugate structures andhydrazinyl-indole compounds used to produce these conjugates. Thepresent disclosure also provides methods of production of suchconjugates, as well as methods of using the same.

Embodiments of the present disclosure include a conjugate that includesat least one modified amino acid residue of formula (I):

wherein

n is 0 or 1;

R₁ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl;

R₂ and R₃ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

X₁, X₂, X₃ and X₄ are each independently selected from C, N, O and S;

X₅ is C;

Y₁, Y₂, Y₃, Y₄ and Y₅ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl;

Q₁ is a bond to either X₄ or X₅, wherein if Q₁ is a bond to X₄, then Y₄is absent, or if Q₁ is a bond to X₅, then Y₅ is absent; and

L is an optional linker,

wherein one of W₁ and W₂ is a polypeptide and the other is a drug or adetectable label.

In some embodiments, Q₁ is a bond to X₄ and Y₄ is absent.

In some embodiments, Q₁ is a bond to X₅ and Y₅ is absent.

In some embodiments, n is 1.

In some embodiments, R₂ and R₃ are each independently selected fromalkyl and substituted alkyl.

In some embodiments, R₂ and R₃ are each methyl.

In some embodiments, X₁, X₂, X₃ and X₄ are each C.

In some embodiments, Y₁, Y₂ and Y₃ are each H, and one of either Y₄ orY₅ is H.

In some embodiments, L is present and includes a group selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl.

In some embodiments, L is present and includes a polymer. In someembodiments, the polymer is a polyethylene glycol.

In some embodiments, the detectable label includes a fluorophore.

In some embodiments, W₁ is the drug or the detectable label, and W₂ isthe polypeptide.

In some embodiments, W₁ is the polypeptide, and W₂ is the drug or thedetectable label.

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (II):

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (IIa):

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (III):

In some embodiments, the conjugate includes at least one modified aminoacid residue of formula (IIIa):

Embodiments of the present disclosure include a compound of formula(IV):

wherein

one of Q₂ and Q₃ is —(CH₂)_(n)NR₃NHR₂ and the other is Y₄;

n is 0 or 1;

R₂ and R₃ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

X₁, X₂, X₃ and X₄ are each independently selected from C, N, O and S;

Y₁, Y₂, Y₃ and Y₄ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl;

L is an optional linker; and

W₁ is selected from a drug, a detectable label and a polypeptide.

In some embodiments, Q₂ —(CH₂)_(n)NR₃NHR₂ and Q₃ is Y₄.

In some embodiments, Q₃ —(CH₂)_(n)NR₃NHR₂ and Q₂ is Y₄.

In some embodiments, n is 1.

In some embodiments, R₂ and R₃ are each independently selected fromalkyl and substituted alkyl.

In some embodiments, R₂ and R₃ are each methyl.

In some embodiments, X₁, X₂, X₃ and X₄ are each C.

In some embodiments, Y₁, Y₂, Y₃ and Y₄ are each H.

In some embodiments, L is present and includes a group selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl.

In some embodiments, L is present and includes a polymer. In someembodiments, the polymer is a polyethylene glycol.

In some embodiments, the detectable label includes a fluorophore.

In some embodiments, the compound is a compound of formula (V):

In some embodiments, the compound is a compound of formula (Va):

In some embodiments, the compound is a compound of formula (VI):

In some embodiments, the compound is a compound of formula (VIa):

Embodiments of the present disclosure include a method of producing aconjugate. The method includes combining in a reaction mixture acompound of formula (IV) and a second compound that includes a reactivegroup, where the combining is under reaction conditions suitable topromote reaction between the compound and the reactive group of thesecond compound to form a conjugate. The method also includes isolatingthe conjugate from the reaction mixture.

In some embodiments, W₁ is the drug or the detectable label, and thesecond compound is the polypeptide.

In some embodiments, W₁ is the polypeptide, and the second compound isthe drug or the detectable label.

In some embodiments, the reactive group includes a reactive aldehydegroup or a reactive ketone group.

In some embodiments, the reaction mixture includes water.

In some embodiments, the reaction mixture has a pH of 7.

In some embodiments, the reaction conditions are at a temperature of 37°C.

Embodiments of the present disclosure include a pharmaceuticalcomposition that includes a conjugate of formula (I) and apharmaceutically acceptable excipient.

Embodiments of the present disclosure include a method of delivering aconjugate to a subject. The method includes administering to the subjectan effective amount of a conjugate of formula (I).

Embodiments of the present disclosure include a method of treating acondition in a subject. The method includes administering to the subjecthaving the condition a therapeutically effective amount of apharmaceutical composition that includes a conjugate of formula (I),where the administering is effective to treat the condition in thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 , panel A, and FIG. 1 , panel B, show reaction schemes for theproduction of a polypeptide conjugate that includes a hydrazinyl-indolecoupling moiety, according to embodiments of the present disclosure.

FIG. 2 , panel A, shows a reaction scheme for the synthesis of afunctionalized detectable label, according to embodiments of the presentdisclosure. FIG. 2 , panel B, shows a schematic of a conjugationreaction of the functionalized detectable label to an antibody,according to embodiments of the present disclosure. FIG. 2 , panel C,shows images of SDS-PAGE gels showing the results of the reaction,according to embodiments of the present disclosure.

FIG. 3 shows images of SDS-PAGE gels showing the stability of anantibody-detectable label conjugate over a 6 day time period, accordingto embodiments of the present disclosure.

FIG. 4 shows a hydrophobic interaction column (HIC) trace ofaldehyde-tagged antibody conjugated to HIPS-6PEG-Maytansine, accordingto embodiments of the present disclosure.

FIG. 5 , panels A and B, show kinetics of condensation reactions ofmodel amine compounds with benzyloxyacetaldehyde, according toembodiments of the present disclosure. FIG. 5 , panel A, shows thechemical structures of a panel of amines used in the experiment. FIG. 5, panel B, shows a graph of percent conversion to product in sodiumcitrate (pH 4.0-5.5) or sodium phosphate (pH 6.0-7.5) buffers. Reactionscontained 50 μM amine and benzyloxyacetaldehyde and proceeded at roomtemperature for 2 h prior to HPLC analysis.

FIG. 6 , panels A-D, show HPLC traces for small molecule kineticsexperiments, showing reaction of Compound 4 (FIG. 6 , panel A), Compound6 (FIG. 6 , panel B), Compound 7 (FIG. 6 , panel C), and Compound 8(FIG. 6 , panel D) with benzyloxyacetaldehyde after 2 h at pH 6.0,according to embodiments of the present disclosure.Benzyloxyacetaldehyde (°) and bromocresol green (*), which was added asan internal standard, are marked on each chromatogram.

FIG. 7 , panels A-D, show fluorescent labeling of aldehyde-bearingproteins with the HIPS ligation and other common aldehyde bioconjugationchemistries, according to embodiments of the present disclosure. FIG. 7, panel A, shows structures of the HIPS ligation reagent (Compound 9),the hydrazide reagent (Compound 10), the Pictet-Spengler ligationreagent (Compound 11), and the aminooxy reagent (Compound 12). Gel scansshow the relative labeling of FGly-MBP (FIG. 7 , panel B), FGly-α-HER2(FIG. 7 , panel C), and N-terminally transaminated Mb with reagents 9-12(FIG. 7 , panel D). In all cases, buffered protein solutions at pH 6.0were treated with 400 μM fluorophore for 2 h at 37° C. prior to analysisby SDS-PAGE.

FIG. 8 shows a graph of MBP-AF488 conjugate hydrolysis over 5 days,according to embodiments of the present disclosure. Human plasmacontaining 10 μg/mL MBP-AF488 conjugate, linked by either anazacarboline or an oxime, was incubated at 37° C. Aliquots takenapproximately 12 h apart were analyzed by ELISA. Error bars representstandard deviation of six replicate samples.

DEFINITIONS

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.

“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

The term “alkane” refers to alkyl group and alkylene group, as definedherein.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl”refers to the groups R′NHR″— where R′ is alkyl group as defined hereinand R″ is alkylene, alkenylene or alkynylene group as defined herein.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkoxyamino” refers to the group —NH-alkoxy, wherein alkoxy isdefined herein.

The term “haloalkoxy” refers to the groups alkyl-O— wherein one or morehydrogen atoms on the alkyl group have been substituted with a halogroup and include, by way of examples, groups such as trifluoromethoxy,and the like.

The term “haloalkyl” refers to a substituted alkyl group as describedabove, wherein one or more hydrogen atoms on the alkyl group have beensubstituted with a halo group. Examples of such groups include, withoutlimitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl,trifluoroethyl and the like.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

“Alkenyl” refers to straight chain or branched hydrocarbyl groups havingfrom 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and havingat least 1 and preferably from 1 to 2 sites of double bond unsaturation.This term includes, by way of example, bi-vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

The term “substituted alkenyl” refers to an alkenyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO— substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO— substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Alkynyloxy” refers to the group —O-alkynyl, wherein alkynyl is asdefined herein. Alkynyloxy includes, by way of example, ethynyloxy,propynyloxy, and the like.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclyl-C(O)—, and substitutedheterocyclyl-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. For example, acylincludes the “acetyl” group CH₃C(O)—

“Acylamino” refers to the groups —NR²⁰C(O)alkyl, —NR²⁰C(O)substitutedalkyl, NR²⁰C(O)cycloalkyl, —NR²⁰C(O)substituted cycloalkyl,—NR²⁰C(O)cycloalkenyl, —NR²⁰C(O)substituted cycloalkenyl,—NR²⁰C(O)alkenyl, —NR²⁰C(O)substituted alkenyl, —NR²⁰C(O)alkynyl,—NR²⁰C(O)substituted alkynyl, —NR²⁰C(O)aryl, —NR²⁰C(O)substituted aryl,—NR²⁰C(O)heteroaryl, —NR²⁰C(O)substituted heteroaryl,—NR²⁰C(O)heterocyclic, and —NR²⁰C(O)substituted heterocyclic, whereinR²⁰ is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonyl” or the term “aminoacyl” refers to the group—C(O)NR²¹R²², wherein R²¹ and R²² independently are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R²¹ and R²² are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonylamino” refers to the group —NR²¹C(O)NR²²R²³ where R²¹,R²², and R²³ are independently selected from hydrogen, alkyl, aryl orcycloalkyl, or where two R groups are joined to form a heterocyclylgroup.

The term “alkoxycarbonylamino” refers to the group —NRC(O)OR where eachR is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, andheterocyclyl are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclyl-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclyl are as defined herein.

“Aminosulfonyl” refers to the group —SO₂NR²¹R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic and where R²¹ and R²²are optionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group and alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Sulfonylamino” refers to the group —NR²¹SO₂R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ andR²² are optionally joined together with the atoms bound thereto to forma heterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 18 carbon atoms having a single ring (such as is present in aphenyl group) or a ring system having multiple condensed rings (examplesof such aromatic ring systems include naphthyl, anthryl and indanyl)which condensed rings may or may not be aromatic, provided that thepoint of attachment is through an atom of an aromatic ring. This termincludes, by way of example, phenyl and naphthyl. Unless otherwiseconstrained by the definition for the aryl substituent, such aryl groupscan optionally be substituted with from 1 to 5 substituents, or from 1to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.

“Aryloxy” refers to the group —O-aryl, wherein aryl is as definedherein, including, by way of example, phenoxy, naphthoxy, and the like,including optionally substituted aryl groups as also defined herein.

“Amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that atleast one R is not hydrogen.

The term “azido” refers to the group —N₃.

“Carboxyl,” “carboxy” or “carboxylate” refers to —CO₂H or salts thereof.

“Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or“carboxylalkyl” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl, —C(O)O-alkynyl,—C(O)O-substituted alkynyl, —C(O)O-aryl, —C(O)O-substituted aryl,—C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-cycloalkenyl,—C(O)O-substituted cycloalkenyl, —C(O)O-heteroaryl, —C(O)O-substitutedheteroaryl, —C(O)O-heterocyclic, and —C(O)O-substituted heterocyclic,wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“(Carboxyl ester)oxy” or “carbonate” refers to the groups—O—C(O)O-alkyl, —O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl,—O—C(O)O-substituted alkenyl, —O—C(O)O— alkynyl, —O—C(O)O-substitutedalkynyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)O— cycloalkyl,—O—C(O)O-substituted cycloalkyl, —O—C(O)O-cycloalkenyl,—O—C(O)O-substituted cycloalkenyl, —O—C(O)O-heteroaryl,—O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclic, and—O—C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Cyano” or “nitrile” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyland the like. Such cycloalkyl groups include, by way of example, singlering structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclooctyl, and the like, or multiple ring structures such asadamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple rings and having at least onedouble bond and preferably from 1 to 2 double bonds.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10carbon atoms having single or multiple rings and having at least onetriple bond.

“Cycloalkoxy” refers to —O-cycloalkyl.

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms,such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected fromthe group consisting of oxygen, nitrogen, and sulfur within the ring.Such heteroaryl groups can have a single ring (such as, pyridinyl,imidazolyl or furyl) or multiple condensed rings in a ring system (forexample as in groups such as, indolizinyl, quinolinyl, benzofuran,benzimidazolyl or benzothienyl), wherein at least one ring within thering system is aromatic and at least one ring within the ring system isaromatic, provided that the point of attachment is through an atom of anaromatic ring. In certain embodiments, the nitrogen and/or sulfur ringatom(s) of the heteroaryl group are optionally oxidized to provide forthe N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes,by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, andfuranyl. Unless otherwise constrained by the definition for theheteroaryl substituent, such heteroaryl groups can be optionallysubstituted with 1 to 5 substituents, or from 1 to 3 substituents,selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substitutedalkoxy, substituted alkenyl, substituted alkynyl, substitutedcycloalkyl, substituted cycloalkenyl, amino, substituted amino,aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl, andtrihalomethyl.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl wherealkylene and heteroaryl are defined herein. This term includes, by wayof example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

“Heteroaryloxy” refers to —O-heteroaryl.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In certainembodiments, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂— moieties.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, or from 1 to 3 substituents, selected from alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl, and fused heterocycle.

“Heterocyclyloxy” refers to the group —O-heterocyclyl.

The term “heterocyclylthio” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein.

The term “hydroxyamino” refers to the group —NHOH.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

“Sulfonyl” refers to the group SO₂-alkyl, SO₂-substituted alkyl,SO₂-alkenyl, SO₂-substituted alkenyl, SO₂-cycloalkyl, SO₂-substitutedcycloalkyl, SO₂-cycloalkenyl, SO₂-substituted cylcoalkenyl, SO₂-aryl,SO₂-substituted aryl, SO₂-heteroaryl, SO₂-substituted heteroaryl,SO₂-heterocyclic, and SO₂-substituted heterocyclic, wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein. Sulfonyl includes, by way of example, methyl-SO₂—, phenyl-SO₂—,and 4-methylphenyl-SO₂—.

“Sulfonyloxy” refers to the group —OSO₂-alkyl, OSO₂-substituted alkyl,OSO₂-alkenyl, OSO₂-substituted alkenyl, OSO₂-cycloalkyl,OSO₂-substituted cycloalkyl, OSO₂-cycloalkenyl, OSO₂-substitutedcylcoalkenyl, OSO₂-aryl, OSO₂-substituted aryl, OSO₂-heteroaryl,OSO₂-substituted heteroaryl, OSO₂-heterocyclic, and OSO₂ substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

The term “aminocarbonyloxy” refers to the group —OC(O)NRR where each Ris independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thioxo” or the term “thioketo” refers to the atom (═S).

“Alkylthio” or the term “thioalkoxy” refers to the group —S-alkyl,wherein alkyl is as defined herein. In certain embodiments, sulfur maybe oxidized to —S(O)—. The sulfoxide may exist as one or morestereoisomers.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined herein including optionally substituted aryl groupsalso defined herein.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined herein including optionallysubstituted aryl groups as also defined herein.

The term “thioheterocyclooxy” refers to the group heterocyclyl-S—wherein the heterocyclyl group is as defined herein including optionallysubstituted heterocyclyl groups as also defined herein.

In addition to the disclosure herein, the term “substituted,” when usedto modify a specified group or radical, can also mean that one or morehydrogen atoms of the specified group or radical are each, independentlyof one another, replaced with the same or different substituent groupsas defined below.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for substituting for one or more hydrogens(any two hydrogens on a single carbon can be replaced with ═O, ═NR⁷⁰,═N—OR⁷⁰, ═N₂ or ═S) on saturated carbon atoms in the specified group orradical are, unless otherwise specified, —R⁶⁰, halo, ═O, —OR⁷⁰, —SR⁷⁰,—NR⁸⁰R⁸⁰, trihalomethyl, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —SO₂R⁷⁰,—SO₂O⁻M⁺, —SO₂OR⁷⁰, —OSO₂R⁷⁰, —OSO₂O⁻M⁺, —OSO₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂,—P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰,—C(O)O⁻M⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰,—OC(O)R⁷⁰, —OC(S)R⁷⁰, —OC(O)O⁻M⁺, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰,—NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ is selected from thegroup consisting of optionally substituted alkyl, cycloalkyl,heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl,heteroaryl and heteroarylalkyl, each R⁷⁰ is independently hydrogen orR⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, two R⁸⁰'s, takentogether with the nitrogen atom to which they are bonded, form a 5-, 6-or 7-membered heterocycloalkyl which may optionally include from 1 to 4of the same or different additional heteroatoms selected from the groupconsisting of O, N and S, of which N may have —H or C1-C3 alkylsubstitution; and each M⁺ is a counter ion with a net single positivecharge. Each M⁺ may independently be, for example, an alkali ion, suchas K⁺, Na⁺, Li⁺; an ammonium ion, such as ⁺N(R⁶⁰)₄; or an alkaline earthion, such as [Ca²⁺]_(0.5), [Mg²⁺]_(0.5), or [Ba²⁺]_(0.5) (“subscript 0.5means that one of the counter ions for such divalent alkali earth ionscan be an ionized form of a compound of the invention and the other atypical counter ion such as chloride, or two ionized compounds disclosedherein can serve as counter ions for such divalent alkali earth ions, ora doubly ionized compound of the invention can serve as the counter ionfor such divalent alkali earth ions). As specific examples, —NR⁸⁰R⁸⁰ ismeant to include —NH₂, —NH-alkyl, N-pyrrolidinyl, N-piperazinyl,4N-methyl-piperazin-1-yl and N-morpholinyl.

In addition to the disclosure herein, substituent groups for hydrogenson unsaturated carbon atoms in “substituted” alkene, alkyne, aryl andheteroaryl groups are, unless otherwise specified, —R⁶⁰, halo, —O⁻M⁺,—OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰, trihalomethyl, —CF₃, —CN, —OCN, —SCN,—NO, —NO₂, —N₃, —SO₂R⁷⁰, —SO₃M⁺, —SO₃R⁷⁰, —OSO₂R⁷⁰, —OSO₃-M⁺, —OSO₃R⁷⁰,—PO₃ ⁻²(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰,—C(NR⁷⁰)R⁷⁰, —CO₂M⁺, —CO₂R⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰,—OC(O)R⁷⁰, —OC(S)R⁷⁰, —OCO₂M⁺, —OCO₂R⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂-M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰,—NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ areas previously defined, provided that in case of substituted alkene oralkyne, the substituents are not —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, or —S⁻M⁺.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for hydrogens on nitrogen atoms in“substituted” heteroalkyl and cycloheteroalkyl groups are, unlessotherwise specified, —R⁶⁰, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰,trihalomethyl, —CF₃, —CN, —NO, —NO₂, —S(O)₂R⁷⁰, —S(O)₂O⁻M⁺, —S(O)₂OR⁷⁰,—OS(O)₂R⁷⁰, —OS(O)₂O⁻M⁺, —OS(O)₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺,—P(O)(OR⁷⁰)(OR⁷⁰), —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —C(O)OR⁷⁰,—C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰,—OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰C(O)OR⁷⁰,—NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and—NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previouslydefined.

In addition to the disclosure herein, in a certain embodiment, a groupthat is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3substituents, 1 or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)—O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (salts withcounterions having acceptable mammalian safety for a given dosageregime). Such salts can be derived from pharmaceutically acceptableinorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers topharmaceutically acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions well known in theart and include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, formate, tartrate, besylate,mesylate, acetate, maleate, oxalate, and the like.

The term “salt thereof” means a compound formed when a proton of an acidis replaced by a cation, such as a metal cation or an organic cation andthe like. Where applicable, the salt is a pharmaceutically acceptablesalt, although this is not required for salts of intermediate compoundsthat are not intended for administration to a patient. By way ofexample, salts of the present compounds include those wherein thecompound is protonated by an inorganic or organic acid to form a cation,with the conjugate base of the inorganic or organic acid as the anioniccomponent of the salt.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.When the solvent is water, the solvate formed is a hydrate.

“Stereoisomer” and “stereoisomers” refer to compounds that have sameatomic connectivity but different atomic arrangement in space.Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers,and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only inelectronic bonding of atoms and/or in the position of a proton, such asenol-keto and imine-enamine tautomers, or the tautomeric forms ofheteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Aperson of ordinary skill in the art would recognize that othertautomeric ring atom arrangements are possible.

It will be appreciated that the term “or a salt or solvate orstereoisomer thereof” is intended to include all permutations of salts,solvates and stereoisomers, such as a solvate of a pharmaceuticallyacceptable salt of a stereoisomer of subject compound.

“Pharmaceutically effective amount” and “therapeutically effectiveamount” refer to an amount of a compound sufficient to treat a specifieddisorder or disease or one or more of its symptoms and/or to prevent theoccurrence of the disease or disorder. In reference to tumorigenicproliferative disorders, a pharmaceutically or therapeutically effectiveamount comprises an amount sufficient to, among other things, cause thetumor to shrink or decrease the growth rate of the tumor.

“Patient” refers to human and non-human subjects, especially mammaliansubjects.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition in a patient, such as amammal (particularly a human) that includes: (a) preventing the diseaseor medical condition from occurring, such as, prophylactic treatment ofa subject; (b) ameliorating the disease or medical condition, such as,eliminating or causing regression of the disease or medical condition ina patient; (c) suppressing the disease or medical condition, for exampleby, slowing or arresting the development of the disease or medicalcondition in a patient; or (d) alleviating a symptom of the disease ormedical condition in a patient.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymeric form of amino acids ofany length. Unless specifically indicated otherwise, “polypeptide,”“peptide,” and “protein” can include genetically coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones. The termincludes fusion proteins, including, but not limited to, fusion proteinswith a heterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, proteins which contain at least oneN-terminal methionine residue (e.g., to facilitate production in arecombinant bacterial host cell); immunologically tagged proteins; andthe like.

“Native amino acid sequence” or “parent amino acid sequence” are usedinterchangeably herein to refer to the amino acid sequence of apolypeptide prior to modification to include a modified amino acidresidue.

The terms “amino acid analog,” “unnatural amino acid,” and the like maybe used interchangeably, and include amino acid-like compounds that aresimilar in structure and/or overall shape to one or more amino acidscommonly found in naturally occurring proteins (e.g., Ala or A, Cys orC, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K,Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S,Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also includenatural amino acids with modified side chains or backbones. Amino acidsalso include naturally occurring amino acids in D-, rather than L-form.In some instances, the amino acid analogs share backbone structures,and/or the side chain structures of one or more natural amino acids,with difference(s) being one or more modified groups in the molecule.Such modification may include, but is not limited to, substitution of anatom (such as N) for a related atom (such as S), addition of a group(such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.),deletion of a group, substitution of a covalent bond (single bond fordouble bond, etc.), or combinations thereof. For example, amino acidanalogs may include α-hydroxy acids, and α-amino acids, and the like.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, and multispecific antibodies (e.g., bispecificantibodies), humanized antibodies, single-chain antibodies, chimericantibodies, antibody fragments (e.g., Fab fragments), and the like. Anantibody is capable of binding a target antigen. (Janeway, C., Travers,P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., GarlandPublishing, New York). A target antigen can have one or more bindingsites, also called epitopes, recognized by complementarity determiningregions (CDRs) formed by one or more variable regions of an antibody.

The term “natural antibody” refers to an antibody in which the heavy andlight chains of the antibody have been made and paired by the immunesystem of a multi-cellular organism. Spleen, lymph nodes, bone marrowand serum are examples of tissues that produce natural antibodies. Forexample, the antibodies produced by the antibody producing cellsisolated from a first animal immunized with an antigen are naturalantibodies.

The term “humanized antibody” or “humanized immunoglobulin” refers to anon-human (e.g., mouse or rabbit) antibody containing one or more aminoacids (in a framework region, a constant region or a CDR, for example)that have been substituted with a correspondingly positioned amino acidfrom a human antibody. In general, humanized antibodies produce areduced immune response in a human host, as compared to a non-humanizedversion of the same antibody. Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). In certain embodiments, framework substitutions areidentified by modeling of the interactions of the CDR and frameworkresidues to identify framework residues important for antigen bindingand sequence comparison to identify unusual framework residues atparticular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann etal., Nature 332:323 (1988)). Additional methods for humanizingantibodies contemplated for use in the present invention are describedin U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417;5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO98/45331 and WO 98/45332. In particular embodiments, a subject rabbitantibody may be humanized according to the methods set forth inUS20040086979 and US20050033031. Accordingly, the antibodies describedabove may be humanized using methods that are well known in the art.

The term “chimeric antibodies” refer to antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to human constant segments, such asgamma 1 and gamma 3. An example of a therapeutic chimeric antibody is ahybrid protein composed of the variable or antigen-binding domain from amouse antibody and the constant or effector domain from a humanantibody, although domains from other mammalian species may be used.

By “genetically-encodable” as used in reference to an amino acidsequence of polypeptide, peptide or protein means that the amino acidsequence is composed of amino acid residues that are capable ofproduction by transcription and translation of a nucleic acid encodingthe amino acid sequence, where transcription and/or translation mayoccur in a cell or in a cell-free in vitro transcription/translationsystem.

The term “control sequences” refers to DNA sequences that facilitateexpression of an operably linked coding sequence in a particularexpression system, e.g. mammalian cell, bacterial cell, cell-freesynthesis, etc. The control sequences that are suitable for prokaryotesystems, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cell systems mayutilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate the initiation of translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. Linking is accomplished by ligation or throughamplification reactions. Synthetic oligonucleotide adaptors or linkersmay be used for linking sequences in accordance with conventionalpractice.

The term “expression cassette” as used herein refers to a segment ofnucleic acid, usually DNA, that can be inserted into a nucleic acid(e.g., by use of restriction sites compatible with ligation into aconstruct of interest or by homologous recombination into a construct ofinterest or into a host cell genome). In general, the nucleic acidsegment comprises a polynucleotide that encodes a polypeptide ofinterest, and the cassette and restriction sites are designed tofacilitate insertion of the cassette in the proper reading frame fortranscription and translation. Expression cassettes can also compriseelements that facilitate expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least 60% free,at least 75% free, at least 80% free, at least 85% free, at least 90%free, at least 95% free, at least 98% free, or more than 98% free, fromother components with which it is naturally associated.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

By “reactive partner” is meant a molecule or molecular moiety thatspecifically reacts with another reactive partner to produce a reactionproduct. Exemplary reactive partners include a cysteine or serine ofsulfatase motif and Formylglycine Generating Enzyme (FGE), which reactto form a reaction product of a converted aldehyde tag containing aformylglycine (fGly) in lieu of cysteine or serine in the motif. Otherexemplary reactive partners include an aldehyde of an fGly residue of aconverted aldehyde tag and an “aldehyde-reactive reactive partner”,which comprises an aldehyde-reactive group and a moiety of interest, andwhich reacts to form a reaction product of a modified aldehyde taggedpolypeptide having the moiety of interest conjugated to the modifiedpolypeptide through a modified fGly residue.

“N-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free amine group, which amine group in non-N-terminus aminoacid residues normally forms part of the covalent backbone of thepolypeptide.

“C-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free carboxyl group, which carboxyl group in non-C-terminusamino acid residues normally forms part of the covalent backbone of thepolypeptide.

By “internal site” as used in referenced to a polypeptide or an aminoacid sequence of a polypeptide means a region of the polypeptide that isnot at the N-terminus or at the C-terminus.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed, to the extent that suchcombinations embrace subject matter that are, for example, compoundsthat are stable compounds (i.e., compounds that can be made, isolated,characterized, and tested for biological activity). In addition, allsub-combinations of the various embodiments and elements thereof (e.g.,elements of the chemical groups listed in the embodiments describingsuch variables) are also specifically embraced by the present inventionand are disclosed herein just as if each and every such sub-combinationwas individually and explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides conjugates (e.g., polypeptideconjugates), hydrazinyl-indole compounds for producing the conjugatesand methods of making and using the same. Embodiments of each aredescribed in more detail in the sections below.

Conjugates

The present disclosure provides conjugates. By “conjugate” is meant afirst moiety that is stably associated with a second moiety. By “stablyassociated” is meant that a moiety is bound to another moiety orstructure under standard conditions. In certain embodiments, the firstand second moieties are bound to each other through one or more covalentbonds.

In certain embodiments, the conjugate is a polypeptide conjugate, whichincludes a polypeptide conjugated to a second moiety. As described inmore detail below, the moiety conjugated to the polypeptide can be anyof a variety of moieties such as, but not limited to, a detectablelabel, a drug, a water-soluble polymer, or a moiety for immobilizationof the polypeptide to a membrane or a surface. The moiety of interestcan be conjugated to the polypeptide at any desired site of thepolypeptide. Thus, the present disclosure provides, for example, amodified polypeptide having a moiety conjugated at a site at or near theC-terminus of the polypeptide. Other examples include a modifiedpolypeptide having a moiety conjugated at a position at or near theN-terminus of the polypeptide. Examples also include a modifiedpolypeptide having a moiety conjugated at a position between theC-terminus and the N-terminus of the polypeptide (e.g., at an internalsite of the polypeptide). Combinations of the above are also possiblewhere the modified polypeptide is conjugated to two or more moieties.

Embodiments of the present disclosure include conjugates where apolypeptide is conjugated to one or more moieties, such as 2 moieties, 3moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9moieties, or 10 or more moieties. The moieties may be conjugated to thepolypeptide at one or more sites in the polypeptide. For example, apolypeptide may be conjugated to one moiety, e.g., one moiety may beconjugated to a single amino acid residue of the polypeptide. In somecases, a first moiety is conjugated to a first amino acid residue of thepolypeptide and a second moiety is conjugated to a second amino acidresidue of the polypeptide. Additional moieties may be conjugated toother amino acid residues of the polypeptide.

The one or more amino acid residues that are conjugated to the one ormore moieties may be naturally occurring amino acids, unnatural aminoacids, or combinations thereof. For instance, the conjugate may includea moiety conjugated to a naturally occurring amino acid residue of thepolypeptide. In other instances, the conjugate may include a moietyconjugated to an unnatural amino acid residue of the polypeptide. One ormore natural or unnatural amino acid residues in the polypeptide may beconjugated to the moiety or moieties as described herein. For example,two (or more) amino acid residues (e.g., natural or unnatural amino acidresidues) in the polypeptide may each be conjugated to a moiety, suchthat multiple sites in the polypeptide are modified.

Although described herein in terms of a polypeptide conjugated to one ormore moieties (e.g., a drug, a detectable label, a polypeptide, etc.),embodiments of the present disclosure also include conjugates where amoiety (e.g., a drug, a detectable label, a polypeptide, etc.) isconjugated to one or more other moieties (e.g., a drug, a detectablelabel, a polypeptide, etc.). For example, a drug may be conjugated toone or more other moieties (e.g., a drug, a detectable label, apolypeptide, etc.), or in other embodiments, a detectable label may beconjugated to one or more other moieties (e.g., a drug, a detectablelabel, a polypeptide, etc.). Thus, for instance, embodiments of thepresent disclosure include, but are not limited to, the following: aconjugate of a polypeptide and a drug; a conjugate of a polypeptide anda detectable label; a conjugate of two or more polypeptides; a conjugateof two or more drugs; a conjugate of two of more detectable labels; aconjugate of a drug and a detectable label; a conjugate of apolypeptide, a drug and a detectable label; a conjugate of a polypeptideand two or more drugs; a conjugate of a polypeptide and two or moredetectable labels; a conjugate of a drug and two or more polypeptides; aconjugate of a detectable label and two or more polypeptides; and thelike.

In certain embodiments, the polypeptide and the moiety of interest areconjugated through a coupling moiety. For example, the polypeptide andthe moiety of interest may each be bound (e.g., covalently bonded) tothe coupling moiety, thus indirectly binding the polypeptide and themoiety of interest together through the coupling moiety. In some cases,the coupling moiety includes a hydrazinyl-indole compound or aderivative of a hydrazinyl-indole compound. For instance, a generalscheme for coupling a moiety of interest to a polypeptide through ahydrazinyl-indole coupling moiety is shown in the general reactionscheme below.

In the reaction scheme above, R may be the moiety of interest conjugatedto the polypeptide. As described herein, the moiety can be any of avariety of moieties such as, but not limited to, a detectable label, adrug, a water-soluble polymer, or a moiety for immobilization of thepolypeptide to a membrane or a surface of a substrate. R′ and R″ mayeach independently be any desired substituent, such as, but not limitedto, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl.

Other hydrazinyl-indole coupling moieties are also possible. Forexample, another general scheme for coupling a moiety of interest to apolypeptide through a hydrazinyl-indole coupling moiety is shown in thegeneral reaction scheme below.

In the reaction scheme above, R may be the moiety of interest conjugatedto the polypeptide. As described above, the moiety can be any of avariety of moieties such as, but not limited to, a detectable label, adrug, a water-soluble polymer, or a moiety for immobilization of thepolypeptide to a membrane or a surface of a substrate. R′ and R″ mayeach independently be any desired substituent, such as, but not limitedto, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. Other coupling moieties are also possible, asshown in the conjugates and compounds described in more detail below.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (I):

wherein

n is 0 or 1;

R₁ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl;

R₂ and R₃ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

X₁, X₂, X₃ and X₄ are each independently selected from C, N, O and S;

X₅ is C;

Y₁, Y₂, Y₃, Y₄ and Y₅ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl;

Q₁ is a bond to either X₄ or X₅, wherein if Q₁ is a bond to X₄, then Y₄is absent, or if Q₁ is a bond to X₅, then Y₅ is absent; and

L is an optional linker,

wherein one of W₁ and W₂ is a polypeptide and the other is a drug or adetectable label.

In certain embodiments, n is 0 or 1. In certain embodiments, n is 0. Incertain embodiments, n is 1.

In certain embodiments, R₁ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₁ is hydrogen. In certain embodiments, R₁ is alkylor substituted alkyl. In certain embodiments, R₁ is alkenyl orsubstituted alkenyl. In certain embodiments, R₁ is alkynyl orsubstituted alkynyl. In certain embodiments, R₁ is aryl or substitutedaryl. In certain embodiments, R₁ is heteroaryl or substitutedheteroaryl. In certain embodiments, R₁ is cycloalkyl or substitutedcycloalkyl. In certain embodiments, R₁ is heterocyclyl or substitutedheterocyclyl.

In certain embodiments, R₂ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₂ is hydrogen. In certain embodiments, R₂ is alkylor substituted alkyl. In certain embodiments, R₂ is alkenyl orsubstituted alkenyl. In certain embodiments, R₂ is alkynyl orsubstituted alkynyl. In certain embodiments, R₂ is alkoxy or substitutedalkoxy. In certain embodiments, R₂ is amino or substituted amino. Incertain embodiments, R₂ is carboxyl or carboxyl ester. In certainembodiments, R₂ is acyl or acyloxy. In certain embodiments, R₂ is acylamino or amino acyl. In certain embodiments, R₂ is alkylamide orsubstituted alkylamide. In certain embodiments, R₂ is sulfonyl. Incertain embodiments, R₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₂ is aryl or substituted aryl. In certainembodiments, R₂ is heteroaryl or substituted heteroaryl. In certainembodiments, R₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₂ is alkyl or substituted alkyl. For example,R₂ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂ ismethyl.

In certain embodiments, R₃ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₃ is hydrogen. In certain embodiments, R₃ is alkylor substituted alkyl. In certain embodiments, R₃ is alkenyl orsubstituted alkenyl. In certain embodiments, R₃ is alkynyl orsubstituted alkynyl. In certain embodiments, R₃ is alkoxy or substitutedalkoxy. In certain embodiments, R₃ is amino or substituted amino. Incertain embodiments, R₃ is carboxyl or carboxyl ester. In certainembodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃ is acylamino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₃ is alkyl or substituted alkyl. For example,R₃ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₃ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₃ ismethyl.

In certain embodiments, R₂ and R₃ are each independently selected fromalkyl and substituted alkyl. For example, R₂ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R₃ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R₂ and R₃ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂and R₃ are each methyl.

In certain embodiments, X₁ is selected from C, N, O and S. In certainembodiments, X₁ is C. In certain embodiments, X₁ is N. In certainembodiments, X₁ is O. In certain embodiments, X₁ is S.

In certain embodiments, X₂ is selected from C, N, O and S. In certainembodiments, X₂ is C. In certain embodiments, X₂ is N. In certainembodiments, X₂ is O. In certain embodiments, X₂ is S.

In certain embodiments, X₃ is selected from C, N, O and S. In certainembodiments, X₃ is C. In certain embodiments, X₃ is N. In certainembodiments, X₃ is O. In certain embodiments, X₃ is S.

In certain embodiments, X₄ is selected from C, N, O and S. In certainembodiments, X₄ is C. In certain embodiments, X₄ is N. In certainembodiments, X₄ is O. In certain embodiments, X₄ is S.

Various combinations of X₁, X₂, X₃ and X₄ are possible. For example, incertain embodiments, each of X₁, X₂, X₃ and X₄ is C. In other instances,three of X₁, X₂, X₃ and X₄ are C and one of X₁, X₂, X₃ and X₄ is N. Inother embodiments, two of X₁, X₂, X₃ and X₄ are C and two of X₁, X₂, X₃and X₄ are N. In other embodiments, one of X₁, X₂, X₃ and X₄ is C andthree of X₁, X₂, X₃ and X₄ is are N. Other combinations of C, N, O and Sare possible for X₁, X₂, X₃ and X₄ as desired.

In certain embodiments, X₅ is C.

In certain embodiments, Y₁ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₁ is hydrogen. In certain embodiments, Y₁ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₁ is F. Incertain embodiments, Y₁ is Cl. In certain embodiments, Y₁ is Br. Incertain embodiments, Y₁ is I. In certain embodiments, Y₁ is alkyl orsubstituted alkyl. In certain embodiments, Y₁ is alkenyl or substitutedalkenyl. In certain embodiments, Y₁ is alkynyl or substituted alkynyl.In certain embodiments, Y₁ is alkoxy or substituted alkoxy. In certainembodiments, Y₁ is amino or substituted amino. In certain embodiments,Y₁ is carboxyl or carboxyl ester. In certain embodiments, Y₁ is acyl oracyloxy. In certain embodiments, Y₁ is acyl amino or amino acyl. Incertain embodiments, Y₁ is alkylamide or substituted alkylamide. Incertain embodiments, Y₁ is sulfonyl. In certain embodiments, Y₁ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₁ is arylor substituted aryl. In certain embodiments, Y₁ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₁ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₁ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₂ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₂ is hydrogen. In certain embodiments, Y₂ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₂ is F. Incertain embodiments, Y₂ is Cl. In certain embodiments, Y₂ is Br. Incertain embodiments, Y₂ is I. In certain embodiments, Y₂ is alkyl orsubstituted alkyl. In certain embodiments, Y₂ is alkenyl or substitutedalkenyl. In certain embodiments, Y₂ is alkynyl or substituted alkynyl.In certain embodiments, Y₂ is alkoxy or substituted alkoxy. In certainembodiments, Y₂ is amino or substituted amino. In certain embodiments,Y₂ is carboxyl or carboxyl ester. In certain embodiments, Y₂ is acyl oracyloxy. In certain embodiments, Y₂ is acyl amino or amino acyl. Incertain embodiments, Y₂ is alkylamide or substituted alkylamide. Incertain embodiments, Y₂ is sulfonyl. In certain embodiments, Y₂ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₂ is arylor substituted aryl. In certain embodiments, Y₂ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₂ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₂ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₃ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₃ is hydrogen. In certain embodiments, Y₃ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₃ is F. Incertain embodiments, Y₃ is Cl. In certain embodiments, Y₃ is Br. Incertain embodiments, Y₃ is I. In certain embodiments, Y₃ is alkyl orsubstituted alkyl. In certain embodiments, Y₃ is alkenyl or substitutedalkenyl. In certain embodiments, Y₃ is alkynyl or substituted alkynyl.In certain embodiments, Y₃ is alkoxy or substituted alkoxy. In certainembodiments, Y₃ is amino or substituted amino. In certain embodiments,Y₃ is carboxyl or carboxyl ester. In certain embodiments, Y₃ is acyl oracyloxy. In certain embodiments, Y₃ is acyl amino or amino acyl. Incertain embodiments, Y₃ is alkylamide or substituted alkylamide. Incertain embodiments, Y₃ is sulfonyl. In certain embodiments, Y₃ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₃ is arylor substituted aryl. In certain embodiments, Y₃ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₃ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₃ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₄ (if present) is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₄ is hydrogen. Incertain embodiments, Y₄ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₄ is F. In certain embodiments, Y₄ is Cl. In certainembodiments, Y₄ is Br. In certain embodiments, Y₄ is I. In certainembodiments, Y₄ is alkyl or substituted alkyl. In certain embodiments,Y₄ is alkenyl or substituted alkenyl. In certain embodiments, Y₄ isalkynyl or substituted alkynyl. In certain embodiments, Y₄ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₄ is amino or substitutedamino. In certain embodiments, Y₄ is carboxyl or carboxyl ester. Incertain embodiments, Y₄ is acyl or acyloxy. In certain embodiments, Y₄is acyl amino or amino acyl. In certain embodiments, Y₄ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₄ is sulfonyl. Incertain embodiments, Y₄ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₄ is aryl or substituted aryl. In certainembodiments, Y₄ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₄ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₄ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, Y₅ (if present) is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₅ is hydrogen. Incertain embodiments, Y₅ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₅ is F. In certain embodiments, Y₅ is Cl. In certainembodiments, Y₅ is Br. In certain embodiments, Y₅ is I. In certainembodiments, Y₅ is alkyl or substituted alkyl. In certain embodiments,Y₅ is alkenyl or substituted alkenyl. In certain embodiments, Y₅ isalkynyl or substituted alkynyl. In certain embodiments, Y₅ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₅ is amino or substitutedamino. In certain embodiments, Y₅ is carboxyl or carboxyl ester. Incertain embodiments, Y₅ is acyl or acyloxy. In certain embodiments, Y₅is acyl amino or amino acyl. In certain embodiments, Y₅ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₅ is sulfonyl. Incertain embodiments, Y₅ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₅ is aryl or substituted aryl. In certainembodiments, Y₅ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₅ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₅ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, Q₁ is a bond to either X₄ or X₅. In certainembodiments, Q₁ is a bond to X₄. In certain embodiments, if Q₁ is a bondto X₄, then Y₄ is absent. In certain embodiments, Q₁ is a bond to X₅. Incertain embodiments, if Q₁ is a bond to X₅, then Y₅ is absent.

In certain embodiments, L is an optional linker. In certain embodiments,L is not present, and thus the nitrogen of the indole ring is directlybonded to W₁. In certain embodiments, L is present, and thus thenitrogen of the indole ring is indirectly bonded to W₁ through thelinker L.

In certain embodiments, L includes a group selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, L includes an alkyl or substituted alkyl group. Incertain embodiments, L includes an alkenyl or substituted alkenyl group.In certain embodiments, L includes an alkynyl or substituted alkynylgroup. In certain embodiments, L includes an alkoxy or substitutedalkoxy group. In certain embodiments, L includes an amino or substitutedamino group. In certain embodiments, L includes a carboxyl or carboxylester group. In certain embodiments, L includes an acyl amino group. Incertain embodiments, L includes an alkylamide or substituted alkylamidegroup. In certain embodiments, L includes an aryl or substituted arylgroup. In certain embodiments, L includes a heteroaryl or substitutedheteroaryl group. In certain embodiments, L includes a cycloalkyl orsubstituted cycloalkyl group. In certain embodiments, L includes aheterocyclyl or substituted heterocyclyl group.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

In certain embodiments, W₁ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₁ is a drug. In certainembodiments, W₁ is a detectable label. In certain embodiments, W₁ is apolypeptide.

In certain embodiments, W₂ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₂ is a drug. In certainembodiments, W₂ is a detectable label. In certain embodiments, W₂ is apolypeptide.

In certain embodiments, one of W₁ and W₂ is a polypeptide and the otheris a drug or a detectable label. In certain embodiments, W₁ is the drugor the detectable label, and W₂ is the polypeptide. In certainembodiments, W₁ is the polypeptide, and W₂ is the drug or the detectablelabel.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (II):

wherein

n is 0 or 1;

R₁ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl;

R₂ and R₃ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

X₁, X₂, X₃ and X₄ are each independently selected from C, N, O and S;

Y₁, Y₂, Y₃ and Y₄ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl;

L is an optional linker; and

wherein one of W₁ and W₂ is a polypeptide and the other is a drug or adetectable label.

In certain embodiments, the substituents for formula (II) are the sameas for formula (I) described above. For example, in certain embodiments,n is 0 or 1. In certain embodiments, n is 0. In certain embodiments, nis 1.

In certain embodiments, R₁ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₁ is hydrogen. In certain embodiments, R₁ is alkylor substituted alkyl. In certain embodiments, R₁ is alkenyl orsubstituted alkenyl. In certain embodiments, R₁ is alkynyl orsubstituted alkynyl. In certain embodiments, R₁ is aryl or substitutedaryl. In certain embodiments, R₁ is heteroaryl or substitutedheteroaryl. In certain embodiments, R₁ is cycloalkyl or substitutedcycloalkyl. In certain embodiments, R₁ is heterocyclyl or substitutedheterocyclyl.

In certain embodiments, R₂ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₂ is hydrogen. In certain embodiments, R₂ is alkylor substituted alkyl. In certain embodiments, R₂ is alkenyl orsubstituted alkenyl. In certain embodiments, R₂ is alkynyl orsubstituted alkynyl. In certain embodiments, R₂ is alkoxy or substitutedalkoxy. In certain embodiments, R₂ is amino or substituted amino. Incertain embodiments, R₂ is carboxyl or carboxyl ester. In certainembodiments, R₂ is acyl or acyloxy. In certain embodiments, R₂ is acylamino or amino acyl. In certain embodiments, R₂ is alkylamide orsubstituted alkylamide. In certain embodiments, R₂ is sulfonyl. Incertain embodiments, R₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₂ is aryl or substituted aryl. In certainembodiments, R₂ is heteroaryl or substituted heteroaryl. In certainembodiments, R₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₂ is alkyl or substituted alkyl. For example,R₂ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂ ismethyl.

In certain embodiments, R₃ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₃ is hydrogen. In certain embodiments, R₃ is alkylor substituted alkyl. In certain embodiments, R₃ is alkenyl orsubstituted alkenyl. In certain embodiments, R₃ is alkynyl orsubstituted alkynyl. In certain embodiments, R₃ is alkoxy or substitutedalkoxy. In certain embodiments, R₃ is amino or substituted amino. Incertain embodiments, R₃ is carboxyl or carboxyl ester. In certainembodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃ is acylamino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₃ is alkyl or substituted alkyl. For example,R₃ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₃ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₃ ismethyl.

In certain embodiments, R₂ and R₃ are each independently selected fromalkyl and substituted alkyl. For example, R₂ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R₃ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R₂ and R₃ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂and R₃ are each methyl.

In certain embodiments, X₁ is selected from C, N, O and S. In certainembodiments, X₁ is C. In certain embodiments, X₁ is N. In certainembodiments, X₁ is O. In certain embodiments, X₁ is S.

In certain embodiments, X₂ is selected from C, N, O and S. In certainembodiments, X₂ is C. In certain embodiments, X₂ is N. In certainembodiments, X₂ is O. In certain embodiments, X₂ is S.

In certain embodiments, X₃ is selected from C, N, O and S. In certainembodiments, X₃ is C. In certain embodiments, X₃ is N. In certainembodiments, X₃ is O. In certain embodiments, X₃ is S.

In certain embodiments, X₄ is selected from C, N, O and S. In certainembodiments, X₄ is C. In certain embodiments, X₄ is N. In certainembodiments, X₄ is O. In certain embodiments, X₄ is S.

Various combinations of X₁, X₂, X₃ and X₄ are possible. For example, incertain embodiments, each of X₁, X₂, X₃ and X₄ is C. In other instances,three of X₁, X₂, X₃ and X₄ are C and one of X₁, X₂, X₃ and X₄ is N. Inother embodiments, two of X₁, X₂, X₃ and X₄ are C and two of X₁, X₂, X₃and X₄ are N. In other embodiments, one of X₁, X₂, X₃ and X₄ is C andthree of X₁, X₂, X₃ and X₄ is are N. Other combinations of C, N, O and Sare possible for X₁, X₂, X₃ and X₄ as desired.

In certain embodiments, Y₁ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₁ is hydrogen. In certain embodiments, Y₁ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₁ is F. Incertain embodiments, Y₁ is Cl. In certain embodiments, Y₁ is Br. Incertain embodiments, Y₁ is I. In certain embodiments, Y₁ is alkyl orsubstituted alkyl. In certain embodiments, Y₁ is alkenyl or substitutedalkenyl. In certain embodiments, Y₁ is alkynyl or substituted alkynyl.In certain embodiments, Y₁ is alkoxy or substituted alkoxy. In certainembodiments, Y₁ is amino or substituted amino. In certain embodiments,Y₁ is carboxyl or carboxyl ester. In certain embodiments, Y₁ is acyl oracyloxy. In certain embodiments, Y₁ is acyl amino or amino acyl. Incertain embodiments, Y₁ is alkylamide or substituted alkylamide. Incertain embodiments, Y₁ is sulfonyl. In certain embodiments, Y₁ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₁ is arylor substituted aryl. In certain embodiments, Y₁ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₁ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₁ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₂ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₂ is hydrogen. In certain embodiments, Y₂ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₂ is F. Incertain embodiments, Y₂ is Cl. In certain embodiments, Y₂ is Br. Incertain embodiments, Y₂ is I. In certain embodiments, Y₂ is alkyl orsubstituted alkyl. In certain embodiments, Y₂ is alkenyl or substitutedalkenyl. In certain embodiments, Y₂ is alkynyl or substituted alkynyl.In certain embodiments, Y₂ is alkoxy or substituted alkoxy. In certainembodiments, Y₂ is amino or substituted amino. In certain embodiments,Y₂ is carboxyl or carboxyl ester. In certain embodiments, Y₂ is acyl oracyloxy. In certain embodiments, Y₂ is acyl amino or amino acyl. Incertain embodiments, Y₂ is alkylamide or substituted alkylamide. Incertain embodiments, Y₂ is sulfonyl. In certain embodiments, Y₂ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₂ is arylor substituted aryl. In certain embodiments, Y₂ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₂ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₂ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₃ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₃ is hydrogen. In certain embodiments, Y₃ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₃ is F. Incertain embodiments, Y₃ is Cl. In certain embodiments, Y₃ is Br. Incertain embodiments, Y₃ is I. In certain embodiments, Y₃ is alkyl orsubstituted alkyl. In certain embodiments, Y₃ is alkenyl or substitutedalkenyl. In certain embodiments, Y₃ is alkynyl or substituted alkynyl.In certain embodiments, Y₃ is alkoxy or substituted alkoxy. In certainembodiments, Y₃ is amino or substituted amino. In certain embodiments,Y₃ is carboxyl or carboxyl ester. In certain embodiments, Y₃ is acyl oracyloxy. In certain embodiments, Y₃ is acyl amino or amino acyl. Incertain embodiments, Y₃ is alkylamide or substituted alkylamide. Incertain embodiments, Y₃ is sulfonyl. In certain embodiments, Y₃ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₃ is arylor substituted aryl. In certain embodiments, Y₃ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₃ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₃ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₄ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₄ is hydrogen. In certain embodiments, Y₄ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₄ is F. Incertain embodiments, Y₄ is Cl. In certain embodiments, Y₄ is Br. Incertain embodiments, Y₄ is I. In certain embodiments, Y₄ is alkyl orsubstituted alkyl. In certain embodiments, Y₄ is alkenyl or substitutedalkenyl. In certain embodiments, Y₄ is alkynyl or substituted alkynyl.In certain embodiments, Y₄ is alkoxy or substituted alkoxy. In certainembodiments, Y₄ is amino or substituted amino. In certain embodiments,Y₄ is carboxyl or carboxyl ester. In certain embodiments, Y₄ is acyl oracyloxy. In certain embodiments, Y₄ is acyl amino or amino acyl. Incertain embodiments, Y₄ is alkylamide or substituted alkylamide. Incertain embodiments, Y₄ is sulfonyl. In certain embodiments, Y₄ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₄ is arylor substituted aryl. In certain embodiments, Y₄ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₄ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₄ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, L is an optional linker. In certain embodiments,L is not present, and thus the nitrogen of the indole ring is directlybonded to W₁. In certain embodiments, L is present, and thus thenitrogen of the indole ring is indirectly bonded to W₁ through thelinker L.

In certain embodiments, L includes a group selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, L includes an alkyl or substituted alkyl group. Incertain embodiments, L includes an alkenyl or substituted alkenyl group.In certain embodiments, L includes an alkynyl or substituted alkynylgroup. In certain embodiments, L includes an alkoxy or substitutedalkoxy group. In certain embodiments, L includes an amino or substitutedamino group. In certain embodiments, L includes a carboxyl or carboxylester group. In certain embodiments, L includes an acyl amino group. Incertain embodiments, L includes an alkylamide or substituted alkylamidegroup. In certain embodiments, L includes an aryl or substituted arylgroup. In certain embodiments, L includes a heteroaryl or substitutedheteroaryl group. In certain embodiments, L includes a cycloalkyl orsubstituted cycloalkyl group. In certain embodiments, L includes aheterocyclyl or substituted heterocyclyl group.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

In certain embodiments, W₁ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₁ is a drug. In certainembodiments, W₁ is a detectable label. In certain embodiments, W₁ is apolypeptide.

In certain embodiments, W₂ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₂ is a drug. In certainembodiments, W₂ is a detectable label. In certain embodiments, W₂ is apolypeptide.

In certain embodiments, one of W₁ and W₂ is a polypeptide and the otheris a drug or a detectable label. In certain embodiments, W₁ is the drugor the detectable label, and W₂ is the polypeptide. In certainembodiments, W₁ is the polypeptide, and W₂ is the drug or the detectablelabel.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (IIa):

In certain embodiments, the substituents in formula (IIa) are asdescribed above for formula (II). For example, in certain embodiments,R₁ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl. In certainembodiments, R₁ is hydrogen. In certain embodiments, R₁ is alkyl orsubstituted alkyl. In certain embodiments, R₁ is alkenyl or substitutedalkenyl. In certain embodiments, R₁ is alkynyl or substituted alkynyl.In certain embodiments, R₁ is aryl or substituted aryl. In certainembodiments, R₁ is heteroaryl or substituted heteroaryl. In certainembodiments, R₁ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₁ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIa), R₂ is selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, R₂ is hydrogen. In certainembodiments, R₂ is alkyl or substituted alkyl. In certain embodiments,R₂ is alkenyl or substituted alkenyl. In certain embodiments, R₂ isalkynyl or substituted alkynyl. In certain embodiments, R₂ is alkoxy orsubstituted alkoxy. In certain embodiments, R₂ is amino or substitutedamino. In certain embodiments, R₂ is carboxyl or carboxyl ester. Incertain embodiments, R₂ is acyl or acyloxy. In certain embodiments, R₂is acyl amino or amino acyl. In certain embodiments, R₂ is alkylamide orsubstituted alkylamide. In certain embodiments, R₂ is sulfonyl. Incertain embodiments, R₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₂ is aryl or substituted aryl. In certainembodiments, R₂ is heteroaryl or substituted heteroaryl. In certainembodiments, R₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIa), R₂ is alkyl or substitutedalkyl. For example, R₂ may be alkyl or substituted alkyl, such as,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl). In some cases, R₂ is methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₂ is methyl.

In certain embodiments of formula (IIa), R₃ is selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, R₃ is hydrogen. In certainembodiments, R₃ is alkyl or substituted alkyl. In certain embodiments,R₃ is alkenyl or substituted alkenyl. In certain embodiments, R₃ isalkynyl or substituted alkynyl. In certain embodiments, R₃ is alkoxy orsubstituted alkoxy. In certain embodiments, R₃ is amino or substitutedamino. In certain embodiments, R₃ is carboxyl or carboxyl ester. Incertain embodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃is acyl amino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIa), R₃ is alkyl or substitutedalkyl. For example, R₃ may be alkyl or substituted alkyl, such as,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl). In some cases, R₃ is methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₃ is methyl.

In certain embodiments of formula (IIa), R₂ and R₃ are eachindependently selected from alkyl and substituted alkyl. For example, R₂may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl), and R₃may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ and R₃ are each independently selected from methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, orthe like. In certain cases, R₂ and R₃ are each methyl.

In certain embodiments of formula (IIa), Y₁ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₁ is hydrogen. Incertain embodiments, Y₁ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₁ is F. In certain embodiments, Y₁ is Cl. In certainembodiments, Y₁ is Br. In certain embodiments, Y₁ is I. In certainembodiments, Y₁ is alkyl or substituted alkyl. In certain embodiments,Y₁ is alkenyl or substituted alkenyl. In certain embodiments, Y₁ isalkynyl or substituted alkynyl. In certain embodiments, Y₁ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₁ is amino or substitutedamino. In certain embodiments, Y₁ is carboxyl or carboxyl ester. Incertain embodiments, Y₁ is acyl or acyloxy. In certain embodiments, Y₁is acyl amino or amino acyl. In certain embodiments, Y₁ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₁ is sulfonyl. Incertain embodiments, Y₁ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₁ is aryl or substituted aryl. In certainembodiments, Y₁ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₁ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₁ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIa), Y₂ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₂ is hydrogen. Incertain embodiments, Y₂ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₂ is F. In certain embodiments, Y₂ is Cl. In certainembodiments, Y₂ is Br. In certain embodiments, Y₂ is I. In certainembodiments, Y₂ is alkyl or substituted alkyl. In certain embodiments,Y₂ is alkenyl or substituted alkenyl. In certain embodiments, Y₂ isalkynyl or substituted alkynyl. In certain embodiments, Y₂ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₂ is amino or substitutedamino. In certain embodiments, Y₂ is carboxyl or carboxyl ester. Incertain embodiments, Y₂ is acyl or acyloxy. In certain embodiments, Y₂is acyl amino or amino acyl. In certain embodiments, Y₂ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₂ is sulfonyl. Incertain embodiments, Y₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₂ is aryl or substituted aryl. In certainembodiments, Y₂ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIa), Y₃ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₃ is hydrogen. Incertain embodiments, Y₃ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₃ is F. In certain embodiments, Y₃ is Cl. In certainembodiments, Y₃ is Br. In certain embodiments, Y₃ is I. In certainembodiments, Y₃ is alkyl or substituted alkyl. In certain embodiments,Y₃ is alkenyl or substituted alkenyl. In certain embodiments, Y₃ isalkynyl or substituted alkynyl. In certain embodiments, Y₃ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₃ is amino or substitutedamino. In certain embodiments, Y₃ is carboxyl or carboxyl ester. Incertain embodiments, Y₃ is acyl or acyloxy. In certain embodiments, Y₃is acyl amino or amino acyl. In certain embodiments, Y₃ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₃ is sulfonyl. Incertain embodiments, Y₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₃ is aryl or substituted aryl. In certainembodiments, Y₃ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIa), Y₄ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₄ is hydrogen. Incertain embodiments, Y₄ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₄ is F. In certain embodiments, Y₄ is Cl. In certainembodiments, Y₄ is Br. In certain embodiments, Y₄ is I. In certainembodiments, Y₄ is alkyl or substituted alkyl. In certain embodiments,Y₄ is alkenyl or substituted alkenyl. In certain embodiments, Y₄ isalkynyl or substituted alkynyl. In certain embodiments, Y₄ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₄ is amino or substitutedamino. In certain embodiments, Y₄ is carboxyl or carboxyl ester. Incertain embodiments, Y₄ is acyl or acyloxy. In certain embodiments, Y₄is acyl amino or amino acyl. In certain embodiments, Y₄ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₄ is sulfonyl. Incertain embodiments, Y₄ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₄ is aryl or substituted aryl. In certainembodiments, Y₄ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₄ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₄ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIa), L is an optional linker. Incertain embodiments, L is not present, and thus the nitrogen of theindole ring is directly bonded to W₁. In certain embodiments, L ispresent, and thus the nitrogen of the indole ring is indirectly bondedto W₁ through the linker L.

In certain embodiments of formula (IIa), L includes a group selectedfrom alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments of formula (IIa), L includes a polymer. Forexample, the polymer may include a polyalkylene glycol and derivativesthereof, including polyethylene glycol, methoxypolyethylene glycol,polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol (e.g., where thehomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers,polyvinylpyrrolidone, combinations thereof, and the like. In certainembodiments, the polymer is a polyalkylene glycol. In certainembodiments, the polymer is a polyethylene glycol.

In certain embodiments of formula (IIa), W₁ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₁ is adrug. In certain embodiments, W₁ is a detectable label. In certainembodiments, W₁ is a polypeptide.

In certain embodiments of formula (IIa), W₂ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₂ is adrug. In certain embodiments, W₂ is a detectable label. In certainembodiments, W₂ is a polypeptide.

In certain embodiments of formula (IIa), one of W₁ and W₂ is apolypeptide and the other is a drug or a detectable label. In certainembodiments, W₁ is the drug or the detectable label, and W₂ is thepolypeptide. In certain embodiments, W₁ is the polypeptide, and W₂ isthe drug or the detectable label.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (IIb):

In certain embodiments, the substituents in formula (IIb) are asdescribed above for formula (II). For example, in certain embodiments, Lis an optional linker. In certain embodiments, L is not present, andthus the nitrogen of the indole ring is directly bonded to W₁. Incertain embodiments, L is present, and thus the nitrogen of the indolering is indirectly bonded to W₁ through the linker L.

In certain embodiments of formula (IIb), L includes a group selectedfrom alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments of formula (IIb), L includes a polymer. Forexample, the polymer may include a polyalkylene glycol and derivativesthereof, including polyethylene glycol, methoxypolyethylene glycol,polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol (e.g., where thehomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers,polyvinylpyrrolidone, combinations thereof, and the like. In certainembodiments, the polymer is a polyalkylene glycol. In certainembodiments, the polymer is a polyethylene glycol.

In certain embodiments of formula (IIb), W₁ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₁ is adrug. In certain embodiments, W₁ is a detectable label. In certainembodiments, W₁ is a polypeptide.

In certain embodiments of formula (IIb), W₂ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₂ is adrug. In certain embodiments, W₂ is a detectable label. In certainembodiments, W₂ is a polypeptide.

In certain embodiments of formula (IIb), one of W₁ and W₂ is apolypeptide and the other is a drug or a detectable label. In certainembodiments, W₁ is the drug or the detectable label, and W₂ is thepolypeptide. In certain embodiments, W₁ is the polypeptide, and W₂ isthe drug or the detectable label.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (III):

wherein

n is 0 or 1;

R₁ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl;

R₂ and R₃ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

X₁, X₂, X₃ and X₄ are each independently selected from C, N, O and S;

Y₁, Y₂, Y₃ and Y₅ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl;

L is an optional linker; and

wherein one of W₁ and W₂ is a polypeptide and the other is a drug or adetectable label.

In certain embodiments, n is 0 or 1. In certain embodiments, n is 0. Incertain embodiments, n is 1.

In certain embodiments, R₁ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₁ is hydrogen. In certain embodiments, R₁ is alkylor substituted alkyl. In certain embodiments, R₁ is alkenyl orsubstituted alkenyl. In certain embodiments, R₁ is alkynyl orsubstituted alkynyl. In certain embodiments, R₁ is aryl or substitutedaryl. In certain embodiments, R₁ is heteroaryl or substitutedheteroaryl. In certain embodiments, R₁ is cycloalkyl or substitutedcycloalkyl. In certain embodiments, R₁ is heterocyclyl or substitutedheterocyclyl.

In certain embodiments, R₂ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₂ is hydrogen. In certain embodiments, R₂ is alkylor substituted alkyl. In certain embodiments, R₂ is alkenyl orsubstituted alkenyl. In certain embodiments, R₂ is alkynyl orsubstituted alkynyl. In certain embodiments, R₂ is alkoxy or substitutedalkoxy. In certain embodiments, R₂ is amino or substituted amino. Incertain embodiments, R₂ is carboxyl or carboxyl ester. In certainembodiments, R₂ is acyl or acyloxy. In certain embodiments, R₂ is acylamino or amino acyl. In certain embodiments, R₂ is alkylamide orsubstituted alkylamide. In certain embodiments, R₂ is sulfonyl. Incertain embodiments, R₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₂ is aryl or substituted aryl. In certainembodiments, R₂ is heteroaryl or substituted heteroaryl. In certainembodiments, R₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₂ is alkyl or substituted alkyl. For example,R₂ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂ ismethyl.

In certain embodiments, R₃ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₃ is hydrogen. In certain embodiments, R₃ is alkylor substituted alkyl. In certain embodiments, R₃ is alkenyl orsubstituted alkenyl. In certain embodiments, R₃ is alkynyl orsubstituted alkynyl. In certain embodiments, R₃ is alkoxy or substitutedalkoxy. In certain embodiments, R₃ is amino or substituted amino. Incertain embodiments, R₃ is carboxyl or carboxyl ester. In certainembodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃ is acylamino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₃ is alkyl or substituted alkyl. For example,R₃ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₃ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₃ ismethyl.

In certain embodiments, R₂ and R₃ are each independently selected fromalkyl and substituted alkyl. For example, R₂ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R₃ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R₂ and R₃ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂and R₃ are each methyl.

In certain embodiments, X₁ is selected from C, N, O and S. In certainembodiments, X₁ is C. In certain embodiments, X₁ is N. In certainembodiments, X₁ is O. In certain embodiments, X₁ is S.

In certain embodiments, X₂ is selected from C, N, O and S. In certainembodiments, X₂ is C. In certain embodiments, X₂ is N. In certainembodiments, X₂ is O. In certain embodiments, X₂ is S.

In certain embodiments, X₃ is selected from C, N, O and S. In certainembodiments, X₃ is C. In certain embodiments, X₃ is N. In certainembodiments, X₃ is O. In certain embodiments, X₃ is S.

In certain embodiments, X₄ is selected from C, N, O and S. In certainembodiments, X₄ is C. In certain embodiments, X₄ is N. In certainembodiments, X₄ is O. In certain embodiments, X₄ is S.

Various combinations of X₁, X₂, X₃ and X₄ are possible. For example, incertain embodiments, each of X₁, X₂, X₃ and X₄ is C. In other instances,three of X₁, X₂, X₃ and X₄ are C and one of X₁, X₂, X₃ and X₄ is N. Inother embodiments, two of X₁, X₂, X₃ and X₄ are C and two of X₁, X₂, X₃and X₄ are N. In other embodiments, one of X₁, X₂, X₃ and X₄ is C andthree of X₁, X₂, X₃ and X₄ is are N. Other combinations of C, N, O and Sare possible for X₁, X₂, X₃ and X₄ as desired.

In certain embodiments, Y₁ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₁ is hydrogen. In certain embodiments, Y₁ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₁ is F. Incertain embodiments, Y₁ is Cl. In certain embodiments, Y₁ is Br. Incertain embodiments, Y₁ is I. In certain embodiments, Y₁ is alkyl orsubstituted alkyl. In certain embodiments, Y₁ is alkenyl or substitutedalkenyl. In certain embodiments, Y₁ is alkynyl or substituted alkynyl.In certain embodiments, Y₁ is alkoxy or substituted alkoxy. In certainembodiments, Y₁ is amino or substituted amino. In certain embodiments,Y₁ is carboxyl or carboxyl ester. In certain embodiments, Y₁ is acyl oracyloxy. In certain embodiments, Y₁ is acyl amino or amino acyl. Incertain embodiments, Y₁ is alkylamide or substituted alkylamide. Incertain embodiments, Y₁ is sulfonyl. In certain embodiments, Y₁ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₁ is arylor substituted aryl. In certain embodiments, Y₁ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₁ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₁ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₂ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₂ is hydrogen. In certain embodiments, Y₂ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₂ is F. Incertain embodiments, Y₂ is Cl. In certain embodiments, Y₂ is Br. Incertain embodiments, Y₂ is I. In certain embodiments, Y₂ is alkyl orsubstituted alkyl. In certain embodiments, Y₂ is alkenyl or substitutedalkenyl. In certain embodiments, Y₂ is alkynyl or substituted alkynyl.In certain embodiments, Y₂ is alkoxy or substituted alkoxy. In certainembodiments, Y₂ is amino or substituted amino. In certain embodiments,Y₂ is carboxyl or carboxyl ester. In certain embodiments, Y₂ is acyl oracyloxy. In certain embodiments, Y₂ is acyl amino or amino acyl. Incertain embodiments, Y₂ is alkylamide or substituted alkylamide. Incertain embodiments, Y₂ is sulfonyl. In certain embodiments, Y₂ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₂ is arylor substituted aryl. In certain embodiments, Y₂ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₂ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₂ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₃ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₃ is hydrogen. In certain embodiments, Y₃ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₃ is F. Incertain embodiments, Y₃ is Cl. In certain embodiments, Y₃ is Br. Incertain embodiments, Y₃ is I. In certain embodiments, Y₃ is alkyl orsubstituted alkyl. In certain embodiments, Y₃ is alkenyl or substitutedalkenyl. In certain embodiments, Y₃ is alkynyl or substituted alkynyl.In certain embodiments, Y₃ is alkoxy or substituted alkoxy. In certainembodiments, Y₃ is amino or substituted amino. In certain embodiments,Y₃ is carboxyl or carboxyl ester. In certain embodiments, Y₃ is acyl oracyloxy. In certain embodiments, Y₃ is acyl amino or amino acyl. Incertain embodiments, Y₃ is alkylamide or substituted alkylamide. Incertain embodiments, Y₃ is sulfonyl. In certain embodiments, Y₃ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₃ is arylor substituted aryl. In certain embodiments, Y₃ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₃ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₃ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₅ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₅ is hydrogen. In certain embodiments, Y₅ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₅ is F. Incertain embodiments, Y₅ is Cl. In certain embodiments, Y₅ is Br. Incertain embodiments, Y₅ is I. In certain embodiments, Y₅ is alkyl orsubstituted alkyl. In certain embodiments, Y₅ is alkenyl or substitutedalkenyl. In certain embodiments, Y₅ is alkynyl or substituted alkynyl.In certain embodiments, Y₅ is alkoxy or substituted alkoxy. In certainembodiments, Y₅ is amino or substituted amino. In certain embodiments,Y₅ is carboxyl or carboxyl ester. In certain embodiments, Y₅ is acyl oracyloxy. In certain embodiments, Y₅ is acyl amino or amino acyl. Incertain embodiments, Y₅ is alkylamide or substituted alkylamide. Incertain embodiments, Y₅ is sulfonyl. In certain embodiments, Y₅ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₅ is arylor substituted aryl. In certain embodiments, Y₅ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₅ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₅ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, L is an optional linker. In certain embodiments,L is not present, and thus the nitrogen of the indole ring is directlybonded to W₁. In certain embodiments, L is present, and thus thenitrogen of the indole ring is indirectly bonded to W₁ through thelinker L.

In certain embodiments, L includes a group selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, L includes an alkyl or substituted alkyl group. Incertain embodiments, L includes an alkenyl or substituted alkenyl group.In certain embodiments, L includes an alkynyl or substituted alkynylgroup. In certain embodiments, L includes an alkoxy or substitutedalkoxy group. In certain embodiments, L includes an amino or substitutedamino group. In certain embodiments, L includes a carboxyl or carboxylester group. In certain embodiments, L includes an acyl amino group. Incertain embodiments, L includes an alkylamide or substituted alkylamidegroup. In certain embodiments, L includes an aryl or substituted arylgroup. In certain embodiments, L includes a heteroaryl or substitutedheteroaryl group. In certain embodiments, L includes a cycloalkyl orsubstituted cycloalkyl group. In certain embodiments, L includes aheterocyclyl or substituted heterocyclyl group.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

In certain embodiments, W₁ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₁ is a drug. In certainembodiments, W₁ is a detectable label. In certain embodiments, W₁ is apolypeptide.

In certain embodiments, W₂ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₂ is a drug. In certainembodiments, W₂ is a detectable label. In certain embodiments, W₂ is apolypeptide.

In certain embodiments, one of W₁ and W₂ is a polypeptide and the otheris a drug or a detectable label. In certain embodiments, W₁ is the drugor the detectable label, and W₂ is the polypeptide. In certainembodiments, W₁ is the polypeptide, and W₂ is the drug or the detectablelabel.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (IIIa):

In certain embodiments, the substituents in formula (IIIa) are asdescribed above for formula (III). For example, in certain embodiments,R₁ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl. In certainembodiments, R₁ is hydrogen. In certain embodiments, R₁ is alkyl orsubstituted alkyl. In certain embodiments, R₁ is alkenyl or substitutedalkenyl. In certain embodiments, R₁ is alkynyl or substituted alkynyl.In certain embodiments, R₁ is aryl or substituted aryl. In certainembodiments, R₁ is heteroaryl or substituted heteroaryl. In certainembodiments, R₁ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₁ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIIa), R₂ is selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, R₂ is hydrogen. In certainembodiments, R₂ is alkyl or substituted alkyl. In certain embodiments,R₂ is alkenyl or substituted alkenyl. In certain embodiments, R₂ isalkynyl or substituted alkynyl. In certain embodiments, R₂ is alkoxy orsubstituted alkoxy. In certain embodiments, R₂ is amino or substitutedamino. In certain embodiments, R₂ is carboxyl or carboxyl ester. Incertain embodiments, R₂ is acyl or acyloxy. In certain embodiments, R₂is acyl amino or amino acyl. In certain embodiments, R₂ is alkylamide orsubstituted alkylamide. In certain embodiments, R₂ is sulfonyl. Incertain embodiments, R₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₂ is aryl or substituted aryl. In certainembodiments, R₂ is heteroaryl or substituted heteroaryl. In certainembodiments, R₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIIa), R₂ is alkyl or substitutedalkyl. For example, R₂ may be alkyl or substituted alkyl, such as,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl). In some cases, R₂ is methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₂ is methyl.

In certain embodiments of formula (IIIa), R₃ is selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, R₃ is hydrogen. In certainembodiments, R₃ is alkyl or substituted alkyl. In certain embodiments,R₃ is alkenyl or substituted alkenyl. In certain embodiments, R₃ isalkynyl or substituted alkynyl. In certain embodiments, R₃ is alkoxy orsubstituted alkoxy. In certain embodiments, R₃ is amino or substitutedamino. In certain embodiments, R₃ is carboxyl or carboxyl ester. Incertain embodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃is acyl amino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIIa), R₃ is alkyl or substitutedalkyl. For example, R₃ may be alkyl or substituted alkyl, such as,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl). In some cases, R₃ is methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₃ is methyl.

In certain embodiments of formula (IIIa), R₂ and R₃ are eachindependently selected from alkyl and substituted alkyl. For example, R₂may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl), and R₃may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ and R₃ are each independently selected from methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, orthe like. In certain cases, R₂ and R₃ are each methyl.

In certain embodiments of formula (IIIa), Y₁ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₁ is hydrogen. Incertain embodiments, Y₁ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₁ is F. In certain embodiments, Y₁ is Cl. In certainembodiments, Y₁ is Br. In certain embodiments, Y₁ is I. In certainembodiments, Y₁ is alkyl or substituted alkyl. In certain embodiments,Y₁ is alkenyl or substituted alkenyl. In certain embodiments, Y₁ isalkynyl or substituted alkynyl. In certain embodiments, Y₁ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₁ is amino or substitutedamino. In certain embodiments, Y₁ is carboxyl or carboxyl ester. Incertain embodiments, Y₁ is acyl or acyloxy. In certain embodiments, Y₁is acyl amino or amino acyl. In certain embodiments, Y₁ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₁ is sulfonyl. Incertain embodiments, Y₁ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₁ is aryl or substituted aryl. In certainembodiments, Y₁ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₁ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₁ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIIa), Y₂ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₂ is hydrogen. Incertain embodiments, Y₂ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₂ is F. In certain embodiments, Y₂ is Cl. In certainembodiments, Y₂ is Br. In certain embodiments, Y₂ is I. In certainembodiments, Y₂ is alkyl or substituted alkyl. In certain embodiments,Y₂ is alkenyl or substituted alkenyl. In certain embodiments, Y₂ isalkynyl or substituted alkynyl. In certain embodiments, Y₂ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₂ is amino or substitutedamino. In certain embodiments, Y₂ is carboxyl or carboxyl ester. Incertain embodiments, Y₂ is acyl or acyloxy. In certain embodiments, Y₂is acyl amino or amino acyl. In certain embodiments, Y₂ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₂ is sulfonyl. Incertain embodiments, Y₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₂ is aryl or substituted aryl. In certainembodiments, Y₂ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIIa), Y₃ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₃ is hydrogen. Incertain embodiments, Y₃ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₃ is F. In certain embodiments, Y₃ is Cl. In certainembodiments, Y₃ is Br. In certain embodiments, Y₃ is I. In certainembodiments, Y₃ is alkyl or substituted alkyl. In certain embodiments,Y₃ is alkenyl or substituted alkenyl. In certain embodiments, Y₃ isalkynyl or substituted alkynyl. In certain embodiments, Y₃ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₃ is amino or substitutedamino. In certain embodiments, Y₃ is carboxyl or carboxyl ester. Incertain embodiments, Y₃ is acyl or acyloxy. In certain embodiments, Y₃is acyl amino or amino acyl. In certain embodiments, Y₃ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₃ is sulfonyl. Incertain embodiments, Y₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₃ is aryl or substituted aryl. In certainembodiments, Y₃ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIIa), Y₅ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₅ is hydrogen. Incertain embodiments, Y₅ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₅ is F. In certain embodiments, Y₅ is Cl. In certainembodiments, Y₅ is Br. In certain embodiments, Y₅ is I. In certainembodiments, Y₅ is alkyl or substituted alkyl. In certain embodiments,Y₅ is alkenyl or substituted alkenyl. In certain embodiments, Y₅ isalkynyl or substituted alkynyl. In certain embodiments, Y₅ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₅ is amino or substitutedamino. In certain embodiments, Y₅ is carboxyl or carboxyl ester. Incertain embodiments, Y₅ is acyl or acyloxy. In certain embodiments, Y₅is acyl amino or amino acyl. In certain embodiments, Y₅ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₅ is sulfonyl. Incertain embodiments, Y₅ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₅ is aryl or substituted aryl. In certainembodiments, Y₅ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₅ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₅ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (IIIa), L is an optional linker. Incertain embodiments, L is not present, and thus the nitrogen of theindole ring is directly bonded to W₁. In certain embodiments, L ispresent, and thus the nitrogen of the indole ring is indirectly bondedto W₁ through the linker L.

In certain embodiments of formula (IIIa), L includes a group selectedfrom alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments of formula (IIIa), L includes a polymer. Forexample, the polymer may include a polyalkylene glycol and derivativesthereof, including polyethylene glycol, methoxypolyethylene glycol,polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol (e.g., where thehomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers,polyvinylpyrrolidone, combinations thereof, and the like. In certainembodiments, the polymer is a polyalkylene glycol. In certainembodiments, the polymer is a polyethylene glycol.

In certain embodiments of formula (IIIa), W₁ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₁ is adrug. In certain embodiments, W₁ is a detectable label. In certainembodiments, W₁ is a polypeptide.

In certain embodiments of formula (IIIa), W₂ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₂ is adrug. In certain embodiments, W₂ is a detectable label. In certainembodiments, W₂ is a polypeptide.

In certain embodiments of formula (IIIa), one of W₁ and W₂ is apolypeptide and the other is a drug or a detectable label. In certainembodiments, W₁ is the drug or the detectable label, and W₂ is thepolypeptide. In certain embodiments, W₁ is the polypeptide, and W₂ isthe drug or the detectable label.

In certain embodiments, the conjugate includes at least one modifiedamino acid residue of formula (IIIb):

In certain embodiments, the substituents in formula (IIIb) are asdescribed above for formula (III). For example, in certain embodiments,L is an optional linker. In certain embodiments, L is not present, andthus the nitrogen of the indole ring is directly bonded to W₁. Incertain embodiments, L is present, and thus the nitrogen of the indolering is indirectly bonded to W₁ through the linker L.

In certain embodiments of formula (IIIb), L includes a group selectedfrom alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments of formula (IIb), L includes a polymer. Forexample, the polymer may include a polyalkylene glycol and derivativesthereof, including polyethylene glycol, methoxypolyethylene glycol,polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol (e.g., where thehomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers,polyvinylpyrrolidone, combinations thereof, and the like. In certainembodiments, the polymer is a polyalkylene glycol. In certainembodiments, the polymer is a polyethylene glycol.

In certain embodiments of formula (IIIb), W₁ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₁ is adrug. In certain embodiments, W₁ is a detectable label. In certainembodiments, W₁ is a polypeptide.

In certain embodiments of formula (IIIb), W₂ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₂ is adrug. In certain embodiments, W₂ is a detectable label. In certainembodiments, W₂ is a polypeptide.

In certain embodiments of formula (IIIb), one of W₁ and W₂ is apolypeptide and the other is a drug or a detectable label. In certainembodiments, W₁ is the drug or the detectable label, and W₂ is thepolypeptide. In certain embodiments, W₁ is the polypeptide, and W₂ isthe drug or the detectable label.

Hydrazinyl-Indole Compounds Useful for Producing Conjugates

The present disclosure provides compounds (e.g., hydrazinyl-indolecompounds) useful for producing the conjugates described herein. Incertain embodiments, the compound may be a coupling moiety useful forconjugation of a polypeptide and a second moiety. For example, thecompound may be bound to the polypeptide and also bound to the secondmoiety, thus indirectly binding the polypeptide and the second moietytogether.

In certain instances, the compound may be a hydrazinyl-indolederivative. Embodiments of the compound include a compound of formula(IV):

wherein

one of Q₂ and Q₃ is —(CH₂)_(n)NR₃NHR₂ and the other is Y₄;

n is 0 or 1;

R₂ and R₃ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

X₁, X₂, X₃ and X₄ are each independently selected from C, N, O and S;

Y₁, Y₂, Y₃ and Y₄ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl;

L is an optional linker; and

W₁ is selected from a drug, a detectable label and a polypeptide.

In certain embodiments, one of Q₂ and Q₃ is —(CH₂)_(n)NR₃NHR₂ and theother is Y₄. In certain embodiments, Q₂ is —(CH₂)_(n)NR₃NHR₂ and Q₃ isY₄. In certain embodiments, Q₃ is —(CH₂)_(n)NR₃NHR₂ and Q₂ is Y₄.

In certain embodiments, n is 0 or 1. In certain embodiments, n is 0. Incertain embodiments, n is 1.

In certain embodiments, R₂ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₂ is hydrogen. In certain embodiments, R₂ is alkylor substituted alkyl. In certain embodiments, R₂ is alkenyl orsubstituted alkenyl. In certain embodiments, R₂ is alkynyl orsubstituted alkynyl. In certain embodiments, R₂ is alkoxy or substitutedalkoxy. In certain embodiments, R₂ is amino or substituted amino. Incertain embodiments, R₂ is carboxyl or carboxyl ester. In certainembodiments, R₂ is acyl or acyloxy. In certain embodiments, R₂ is acylamino or amino acyl. In certain embodiments, R₂ is alkylamide orsubstituted alkylamide. In certain embodiments, R₂ is sulfonyl. Incertain embodiments, R₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₂ is aryl or substituted aryl. In certainembodiments, R₂ is heteroaryl or substituted heteroaryl. In certainembodiments, R₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₂ is alkyl or substituted alkyl. For example,R₂ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂ ismethyl.

In certain embodiments, R₃ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₃ is hydrogen. In certain embodiments, R₃ is alkylor substituted alkyl. In certain embodiments, R₃ is alkenyl orsubstituted alkenyl. In certain embodiments, R₃ is alkynyl orsubstituted alkynyl. In certain embodiments, R₃ is alkoxy or substitutedalkoxy. In certain embodiments, R₃ is amino or substituted amino. Incertain embodiments, R₃ is carboxyl or carboxyl ester. In certainembodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃ is acylamino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₃ is alkyl or substituted alkyl. For example,R₃ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₃ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₃ ismethyl.

In certain embodiments, R₂ and R₃ are each independently selected fromalkyl and substituted alkyl. For example, R₂ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R₃ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R₂ and R₃ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂and R₃ are each methyl.

In certain embodiments, X₁ is selected from C, N, O and S. In certainembodiments, X₁ is C. In certain embodiments, X₁ is N. In certainembodiments, X₁ is O. In certain embodiments, X₁ is S.

In certain embodiments, X₂ is selected from C, N, O and S. In certainembodiments, X₂ is C. In certain embodiments, X₂ is N. In certainembodiments, X₂ is O. In certain embodiments, X₂ is S.

In certain embodiments, X₃ is selected from C, N, O and S. In certainembodiments, X₃ is C. In certain embodiments, X₃ is N. In certainembodiments, X₃ is O. In certain embodiments, X₃ is S.

In certain embodiments, X₄ is selected from C, N, O and S. In certainembodiments, X₄ is C. In certain embodiments, X₄ is N. In certainembodiments, X₄ is O. In certain embodiments, X₄ is S.

Various combinations of X₁, X₂, X₃ and X₄ are possible. For example, incertain embodiments, each of X₁, X₂, X₃ and X₄ is C. In other instances,three of X₁, X₂, X₃ and X₄ are C and one of X₁, X₂, X₃ and X₄ is N. Inother embodiments, two of X₁, X₂, X₃ and X₄ are C and two of X₁, X₂, X₃and X₄ are N. In other embodiments, one of X₁, X₂, X₃ and X₄ is C andthree of X₁, X₂, X₃ and X₄ is are N. Other combinations of C, N, O and Sare possible for X₁, X₂, X₃ and X₄ as desired.

In certain embodiments, Y₁ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₁ is hydrogen. In certain embodiments, Y₁ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₁ is F. Incertain embodiments, Y₁ is Cl. In certain embodiments, Y₁ is Br. Incertain embodiments, Y₁ is I. In certain embodiments, Y₁ is alkyl orsubstituted alkyl. In certain embodiments, Y₁ is alkenyl or substitutedalkenyl. In certain embodiments, Y₁ is alkynyl or substituted alkynyl.In certain embodiments, Y₁ is alkoxy or substituted alkoxy. In certainembodiments, Y₁ is amino or substituted amino. In certain embodiments,Y₁ is carboxyl or carboxyl ester. In certain embodiments, Y₁ is acyl oracyloxy. In certain embodiments, Y₁ is acyl amino or amino acyl. Incertain embodiments, Y₁ is alkylamide or substituted alkylamide. Incertain embodiments, Y₁ is sulfonyl. In certain embodiments, Y₁ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₁ is arylor substituted aryl. In certain embodiments, Y₁ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₁ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₁ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₂ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₂ is hydrogen. In certain embodiments, Y₂ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₂ is F. Incertain embodiments, Y₂ is Cl. In certain embodiments, Y₂ is Br. Incertain embodiments, Y₂ is I. In certain embodiments, Y₂ is alkyl orsubstituted alkyl. In certain embodiments, Y₂ is alkenyl or substitutedalkenyl. In certain embodiments, Y₂ is alkynyl or substituted alkynyl.In certain embodiments, Y₂ is alkoxy or substituted alkoxy. In certainembodiments, Y₂ is amino or substituted amino. In certain embodiments,Y₂ is carboxyl or carboxyl ester. In certain embodiments, Y₂ is acyl oracyloxy. In certain embodiments, Y₂ is acyl amino or amino acyl. Incertain embodiments, Y₂ is alkylamide or substituted alkylamide. Incertain embodiments, Y₂ is sulfonyl. In certain embodiments, Y₂ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₂ is arylor substituted aryl. In certain embodiments, Y₂ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₂ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₂ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₃ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₃ is hydrogen. In certain embodiments, Y₃ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₃ is F. Incertain embodiments, Y₃ is Cl. In certain embodiments, Y₃ is Br. Incertain embodiments, Y₃ is I. In certain embodiments, Y₃ is alkyl orsubstituted alkyl. In certain embodiments, Y₃ is alkenyl or substitutedalkenyl. In certain embodiments, Y₃ is alkynyl or substituted alkynyl.In certain embodiments, Y₃ is alkoxy or substituted alkoxy. In certainembodiments, Y₃ is amino or substituted amino. In certain embodiments,Y₃ is carboxyl or carboxyl ester. In certain embodiments, Y₃ is acyl oracyloxy. In certain embodiments, Y₃ is acyl amino or amino acyl. Incertain embodiments, Y₃ is alkylamide or substituted alkylamide. Incertain embodiments, Y₃ is sulfonyl. In certain embodiments, Y₃ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₃ is arylor substituted aryl. In certain embodiments, Y₃ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₃ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₃ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₄ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₄ is hydrogen. In certain embodiments, Y₄ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₄ is F. Incertain embodiments, Y₄ is Cl. In certain embodiments, Y₄ is Br. Incertain embodiments, Y₄ is I. In certain embodiments, Y₄ is alkyl orsubstituted alkyl. In certain embodiments, Y₄ is alkenyl or substitutedalkenyl. In certain embodiments, Y₄ is alkynyl or substituted alkynyl.In certain embodiments, Y₄ is alkoxy or substituted alkoxy. In certainembodiments, Y₄ is amino or substituted amino. In certain embodiments,Y₄ is carboxyl or carboxyl ester. In certain embodiments, Y₄ is acyl oracyloxy. In certain embodiments, Y₄ is acyl amino or amino acyl. Incertain embodiments, Y₄ is alkylamide or substituted alkylamide. Incertain embodiments, Y₄ is sulfonyl. In certain embodiments, Y₄ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₄ is arylor substituted aryl. In certain embodiments, Y₄ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₄ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₄ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, L is an optional linker. In certain embodiments,L is not present, and thus the nitrogen of the indole ring is directlybonded to W₁. In certain embodiments, L is present, and thus thenitrogen of the indole ring is indirectly bonded to W₁ through thelinker L.

In certain embodiments, L includes a group selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, L includes an alkyl or substituted alkyl group. Incertain embodiments, L includes an alkenyl or substituted alkenyl group.In certain embodiments, L includes an alkynyl or substituted alkynylgroup. In certain embodiments, L includes an alkoxy or substitutedalkoxy group. In certain embodiments, L includes an amino or substitutedamino group. In certain embodiments, L includes a carboxyl or carboxylester group. In certain embodiments, L includes an acyl amino group. Incertain embodiments, L includes an alkylamide or substituted alkylamidegroup. In certain embodiments, L includes an aryl or substituted arylgroup. In certain embodiments, L includes a heteroaryl or substitutedheteroaryl group. In certain embodiments, L includes a cycloalkyl orsubstituted cycloalkyl group. In certain embodiments, L includes aheterocyclyl or substituted heterocyclyl group.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

In certain embodiments, W₁ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₁ is a drug. In certainembodiments, W₁ is a detectable label. In certain embodiments, W₁ is apolypeptide.

Embodiments of the compound include a compound of formula (V):

wherein

n is 0 or 1;

R₂ and R₃ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

X₁, X₂, X₃ and X₄ are each independently selected from C, N, O and S;

Y₁, Y₂, Y₃ and Y₄ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl;

L is an optional linker; and

W₁ is selected from a drug, a detectable label and a polypeptide.

In certain embodiments, the substituents for formula (V) are the same asfor formula (IV) described above. For example, in certain embodiments, nis 0 or 1. In certain embodiments, n is 0. In certain embodiments, n is1.

In certain embodiments, R₂ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₂ is hydrogen. In certain embodiments, R₂ is alkylor substituted alkyl. In certain embodiments, R₂ is alkenyl orsubstituted alkenyl. In certain embodiments, R₂ is alkynyl orsubstituted alkynyl. In certain embodiments, R₂ is alkoxy or substitutedalkoxy. In certain embodiments, R₂ is amino or substituted amino. Incertain embodiments, R₂ is carboxyl or carboxyl ester. In certainembodiments, R₂ is acyl or acyloxy. In certain embodiments, R₂ is acylamino or amino acyl. In certain embodiments, R₂ is alkylamide orsubstituted alkylamide. In certain embodiments, R₂ is sulfonyl. Incertain embodiments, R₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₂ is aryl or substituted aryl. In certainembodiments, R₂ is heteroaryl or substituted heteroaryl. In certainembodiments, R₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₂ is alkyl or substituted alkyl. For example,R₂ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂ ismethyl.

In certain embodiments, R₃ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₃ is hydrogen. In certain embodiments, R₃ is alkylor substituted alkyl. In certain embodiments, R₃ is alkenyl orsubstituted alkenyl. In certain embodiments, R₃ is alkynyl orsubstituted alkynyl. In certain embodiments, R₃ is alkoxy or substitutedalkoxy. In certain embodiments, R₃ is amino or substituted amino. Incertain embodiments, R₃ is carboxyl or carboxyl ester. In certainembodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃ is acylamino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₃ is alkyl or substituted alkyl. For example,R₃ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₃ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₃ ismethyl.

In certain embodiments, R₂ and R₃ are each independently selected fromalkyl and substituted alkyl. For example, R₂ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R₃ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R₂ and R₃ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂and R₃ are each methyl.

In certain embodiments, X₁ is selected from C, N, O and S. In certainembodiments, X₁ is C. In certain embodiments, X₁ is N. In certainembodiments, X₁ is O. In certain embodiments, X₁ is S.

In certain embodiments, X₂ is selected from C, N, O and S. In certainembodiments, X₂ is C. In certain embodiments, X₂ is N. In certainembodiments, X₂ is O. In certain embodiments, X₂ is S.

In certain embodiments, X₃ is selected from C, N, O and S. In certainembodiments, X₃ is C. In certain embodiments, X₃ is N. In certainembodiments, X₃ is O. In certain embodiments, X₃ is S.

In certain embodiments, X₄ is selected from C, N, O and S. In certainembodiments, X₄ is C. In certain embodiments, X₄ is N. In certainembodiments, X₄ is O. In certain embodiments, X₄ is S.

Various combinations of X₁, X₂, X₃ and X₄ are possible. For example, incertain embodiments, each of X₁, X₂, X₃ and X₄ is C. In other instances,three of X₁, X₂, X₃ and X₄ are C and one of X₁, X₂, X₃ and X₄ is N. Inother embodiments, two of X₁, X₂, X₃ and X₄ are C and two of X₁, X₂, X₃and X₄ are N. In other embodiments, one of X₁, X₂, X₃ and X₄ is C andthree of X₁, X₂, X₃ and X₄ is are N. Other combinations of C, N, O and Sare possible for X₁, X₂, X₃ and X₄ as desired.

In certain embodiments, Y₁ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₁ is hydrogen. In certain embodiments, Y₁ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₁ is F. Incertain embodiments, Y₁ is Cl. In certain embodiments, Y₁ is Br. Incertain embodiments, Y₁ is I. In certain embodiments, Y₁ is alkyl orsubstituted alkyl. In certain embodiments, Y₁ is alkenyl or substitutedalkenyl. In certain embodiments, Y₁ is alkynyl or substituted alkynyl.In certain embodiments, Y₁ is alkoxy or substituted alkoxy. In certainembodiments, Y₁ is amino or substituted amino. In certain embodiments,Y₁ is carboxyl or carboxyl ester. In certain embodiments, Y₁ is acyl oracyloxy. In certain embodiments, Y₁ is acyl amino or amino acyl. Incertain embodiments, Y₁ is alkylamide or substituted alkylamide. Incertain embodiments, Y₁ is sulfonyl. In certain embodiments, Y₁ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₁ is arylor substituted aryl. In certain embodiments, Y₁ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₁ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₁ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₂ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₂ is hydrogen. In certain embodiments, Y₂ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₂ is F. Incertain embodiments, Y₂ is Cl. In certain embodiments, Y₂ is Br. Incertain embodiments, Y₂ is I. In certain embodiments, Y₂ is alkyl orsubstituted alkyl. In certain embodiments, Y₂ is alkenyl or substitutedalkenyl. In certain embodiments, Y₂ is alkynyl or substituted alkynyl.In certain embodiments, Y₂ is alkoxy or substituted alkoxy. In certainembodiments, Y₂ is amino or substituted amino. In certain embodiments,Y₂ is carboxyl or carboxyl ester. In certain embodiments, Y₂ is acyl oracyloxy. In certain embodiments, Y₂ is acyl amino or amino acyl. Incertain embodiments, Y₂ is alkylamide or substituted alkylamide. Incertain embodiments, Y₂ is sulfonyl. In certain embodiments, Y₂ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₂ is arylor substituted aryl. In certain embodiments, Y₂ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₂ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₂ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₃ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₃ is hydrogen. In certain embodiments, Y₃ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₃ is F. Incertain embodiments, Y₃ is Cl. In certain embodiments, Y₃ is Br. Incertain embodiments, Y₃ is I. In certain embodiments, Y₃ is alkyl orsubstituted alkyl. In certain embodiments, Y₃ is alkenyl or substitutedalkenyl. In certain embodiments, Y₃ is alkynyl or substituted alkynyl.In certain embodiments, Y₃ is alkoxy or substituted alkoxy. In certainembodiments, Y₃ is amino or substituted amino. In certain embodiments,Y₃ is carboxyl or carboxyl ester. In certain embodiments, Y₃ is acyl oracyloxy. In certain embodiments, Y₃ is acyl amino or amino acyl. Incertain embodiments, Y₃ is alkylamide or substituted alkylamide. Incertain embodiments, Y₃ is sulfonyl. In certain embodiments, Y₃ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₃ is arylor substituted aryl. In certain embodiments, Y₃ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₃ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₃ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₄ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₄ is hydrogen. In certain embodiments, Y₄ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₄ is F. Incertain embodiments, Y₄ is Cl. In certain embodiments, Y₄ is Br. Incertain embodiments, Y₄ is I. In certain embodiments, Y₄ is alkyl orsubstituted alkyl. In certain embodiments, Y₄ is alkenyl or substitutedalkenyl. In certain embodiments, Y₄ is alkynyl or substituted alkynyl.In certain embodiments, Y₄ is alkoxy or substituted alkoxy. In certainembodiments, Y₄ is amino or substituted amino. In certain embodiments,Y₄ is carboxyl or carboxyl ester. In certain embodiments, Y₄ is acyl oracyloxy. In certain embodiments, Y₄ is acyl amino or amino acyl. Incertain embodiments, Y₄ is alkylamide or substituted alkylamide. Incertain embodiments, Y₄ is sulfonyl. In certain embodiments, Y₄ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₄ is arylor substituted aryl. In certain embodiments, Y₄ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₄ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₄ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, L is an optional linker. In certain embodiments,L is not present, and thus the nitrogen of the indole ring is directlybonded to W₁. In certain embodiments, L is present, and thus thenitrogen of the indole ring is indirectly bonded to W₁ through thelinker L.

In certain embodiments, L includes a group selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, L includes an alkyl or substituted alkyl group. Incertain embodiments, L includes an alkenyl or substituted alkenyl group.In certain embodiments, L includes an alkynyl or substituted alkynylgroup. In certain embodiments, L includes an alkoxy or substitutedalkoxy group. In certain embodiments, L includes an amino or substitutedamino group. In certain embodiments, L includes a carboxyl or carboxylester group. In certain embodiments, L includes an acyl amino group. Incertain embodiments, L includes an alkylamide or substituted alkylamidegroup. In certain embodiments, L includes an aryl or substituted arylgroup. In certain embodiments, L includes a heteroaryl or substitutedheteroaryl group. In certain embodiments, L includes a cycloalkyl orsubstituted cycloalkyl group. In certain embodiments, L includes aheterocyclyl or substituted heterocyclyl group.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

In certain embodiments, W₁ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₁ is a drug. In certainembodiments, W₁ is a detectable label. In certain embodiments, W₁ is apolypeptide.

Embodiments of the compound include a compound of formula (Va):

In certain embodiments, the substituents in formula (Va) are asdescribed above for formula (V). For example, in certain embodiments, R₂is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments, R₂is hydrogen. In certain embodiments, R₂ is alkyl or substituted alkyl.In certain embodiments, R₂ is alkenyl or substituted alkenyl. In certainembodiments, R₂ is alkynyl or substituted alkynyl. In certainembodiments, R₂ is alkoxy or substituted alkoxy. In certain embodiments,R₂ is amino or substituted amino. In certain embodiments, R₂ is carboxylor carboxyl ester. In certain embodiments, R₂ is acyl or acyloxy. Incertain embodiments, R₂ is acyl amino or amino acyl. In certainembodiments, R₂ is alkylamide or substituted alkylamide. In certainembodiments, R₂ is sulfonyl. In certain embodiments, R₂ is thioalkoxy orsubstituted thioalkoxy. In certain embodiments, R₂ is aryl orsubstituted aryl. In certain embodiments, R₂ is heteroaryl orsubstituted heteroaryl. In certain embodiments, R₂ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, R₂ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments of formula (Va), R₂ is alkyl or substitutedalkyl. For example, R₂ may be alkyl or substituted alkyl, such as,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl). In some cases, R₂ is methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₂ is methyl.

In certain embodiments of formula (Va), R₃ is selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, R₃ is hydrogen. In certainembodiments, R₃ is alkyl or substituted alkyl. In certain embodiments,R₃ is alkenyl or substituted alkenyl. In certain embodiments, R₃ isalkynyl or substituted alkynyl. In certain embodiments, R₃ is alkoxy orsubstituted alkoxy. In certain embodiments, R₃ is amino or substitutedamino. In certain embodiments, R₃ is carboxyl or carboxyl ester. Incertain embodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃is acyl amino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (Va), R₃ is alkyl or substitutedalkyl. For example, R₃ may be alkyl or substituted alkyl, such as,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl). In some cases, R₃ is methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₃ is methyl.

In certain embodiments of formula (Va), R₂ and R₃ are each independentlyselected from alkyl and substituted alkyl. For example, R₂ may be alkylor substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl(e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl), and R₃ may be alkyl orsubstituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl(e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). In some cases, R₂ and R₃are each independently selected from methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₂ and R₃ are each methyl.

In certain embodiments of formula (Va), Y₁ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₁ is hydrogen. Incertain embodiments, Y₁ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₁ is F. In certain embodiments, Y₁ is Cl. In certainembodiments, Y₁ is Br. In certain embodiments, Y₁ is I. In certainembodiments, Y₁ is alkyl or substituted alkyl. In certain embodiments,Y₁ is alkenyl or substituted alkenyl. In certain embodiments, Y₁ isalkynyl or substituted alkynyl. In certain embodiments, Y₁ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₁ is amino or substitutedamino. In certain embodiments, Y₁ is carboxyl or carboxyl ester. Incertain embodiments, Y₁ is acyl or acyloxy. In certain embodiments, Y₁is acyl amino or amino acyl. In certain embodiments, Y₁ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₁ is sulfonyl. Incertain embodiments, Y₁ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₁ is aryl or substituted aryl. In certainembodiments, Y₁ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₁ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₁ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (Va), Y₂ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₂ is hydrogen. Incertain embodiments, Y₂ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₂ is F. In certain embodiments, Y₂ is Cl. In certainembodiments, Y₂ is Br. In certain embodiments, Y₂ is I. In certainembodiments, Y₂ is alkyl or substituted alkyl. In certain embodiments,Y₂ is alkenyl or substituted alkenyl. In certain embodiments, Y₂ isalkynyl or substituted alkynyl. In certain embodiments, Y₂ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₂ is amino or substitutedamino. In certain embodiments, Y₂ is carboxyl or carboxyl ester. Incertain embodiments, Y₂ is acyl or acyloxy. In certain embodiments, Y₂is acyl amino or amino acyl. In certain embodiments, Y₂ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₂ is sulfonyl. Incertain embodiments, Y₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₂ is aryl or substituted aryl. In certainembodiments, Y₂ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (Va), Y₃ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₃ is hydrogen. Incertain embodiments, Y₃ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₃ is F. In certain embodiments, Y₃ is Cl. In certainembodiments, Y₃ is Br. In certain embodiments, Y₃ is I. In certainembodiments, Y₃ is alkyl or substituted alkyl. In certain embodiments,Y₃ is alkenyl or substituted alkenyl. In certain embodiments, Y₃ isalkynyl or substituted alkynyl. In certain embodiments, Y₃ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₃ is amino or substitutedamino. In certain embodiments, Y₃ is carboxyl or carboxyl ester. Incertain embodiments, Y₃ is acyl or acyloxy. In certain embodiments, Y₃is acyl amino or amino acyl. In certain embodiments, Y₃ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₃ is sulfonyl. Incertain embodiments, Y₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₃ is aryl or substituted aryl. In certainembodiments, Y₃ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (Va), Y₄ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₄ is hydrogen. Incertain embodiments, Y₄ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₄ is F. In certain embodiments, Y₄ is Cl. In certainembodiments, Y₄ is Br. In certain embodiments, Y₄ is I. In certainembodiments, Y₄ is alkyl or substituted alkyl. In certain embodiments,Y₄ is alkenyl or substituted alkenyl. In certain embodiments, Y₄ isalkynyl or substituted alkynyl. In certain embodiments, Y₄ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₄ is amino or substitutedamino. In certain embodiments, Y₄ is carboxyl or carboxyl ester. Incertain embodiments, Y₄ is acyl or acyloxy. In certain embodiments, Y₄is acyl amino or amino acyl. In certain embodiments, Y₄ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₄ is sulfonyl. Incertain embodiments, Y₄ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₄ is aryl or substituted aryl. In certainembodiments, Y₄ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₄ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₄ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (Va), L is an optional linker. Incertain embodiments, L is not present, and thus the nitrogen of theindole ring is directly bonded to W₁. In certain embodiments, L ispresent, and thus the nitrogen of the indole ring is indirectly bondedto W₁ through the linker L.

In certain embodiments of formula (Va), L includes a group selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments of formula (Va), L includes a polymer. Forexample, the polymer may include a polyalkylene glycol and derivativesthereof, including polyethylene glycol, methoxypolyethylene glycol,polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol (e.g., where thehomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers,polyvinylpyrrolidone, combinations thereof, and the like. In certainembodiments, the polymer is a polyalkylene glycol. In certainembodiments, the polymer is a polyethylene glycol.

In certain embodiments of formula (Va), W₁ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₁ is adrug. In certain embodiments, W₁ is a detectable label. In certainembodiments, W₁ is a polypeptide.

Embodiments of the compound include a compound of formula (Vb):

In certain embodiments, the substituents in formula (Vb) are asdescribed above for formula (V). For example, in certain embodiments, Lis an optional linker. In certain embodiments, L is not present, andthus the nitrogen of the indole ring is directly bonded to W₁. Incertain embodiments, L is present, and thus the nitrogen of the indolering is indirectly bonded to W₁ through the linker L.

In certain embodiments of formula (Vb), L includes a group selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments of formula (Vb), L includes a polymer. Forexample, the polymer may include a polyalkylene glycol and derivativesthereof, including polyethylene glycol, methoxypolyethylene glycol,polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol (e.g., where thehomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers,polyvinylpyrrolidone, combinations thereof, and the like. In certainembodiments, the polymer is a polyalkylene glycol. In certainembodiments, the polymer is a polyethylene glycol.

In certain embodiments of formula (Vb), W₁ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₁ is adrug. In certain embodiments, W₁ is a detectable label. In certainembodiments, W₁ is a polypeptide.

In certain embodiments, the compound may be a hydrazinyl-indolederivative of formula (VI):

wherein

n is 0 or 1;

R₂ and R₃ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

X₁, X₂, X₃ and X₄ are each independently selected from C, N, O and S;

Y₁, Y₂, Y₃ and Y₄ are each independently selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl;

L is an optional linker; and

W₁ is selected from a drug, a detectable label and a polypeptide.

In certain embodiments, the substituents in formula (VI) are asdescribed above for formula (IV). For example, in certain embodiments, nis 0 or 1. In certain embodiments, n is 0. In certain embodiments, n is1.

In certain embodiments, R₂ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₂ is hydrogen. In certain embodiments, R₂ is alkylor substituted alkyl. In certain embodiments, R₂ is alkenyl orsubstituted alkenyl. In certain embodiments, R₂ is alkynyl orsubstituted alkynyl. In certain embodiments, R₂ is alkoxy or substitutedalkoxy. In certain embodiments, R₂ is amino or substituted amino. Incertain embodiments, R₂ is carboxyl or carboxyl ester. In certainembodiments, R₂ is acyl or acyloxy. In certain embodiments, R₂ is acylamino or amino acyl. In certain embodiments, R₂ is alkylamide orsubstituted alkylamide. In certain embodiments, R₂ is sulfonyl. Incertain embodiments, R₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₂ is aryl or substituted aryl. In certainembodiments, R₂ is heteroaryl or substituted heteroaryl. In certainembodiments, R₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₂ is alkyl or substituted alkyl. For example,R₂ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂ ismethyl.

In certain embodiments, R₃ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R₃ is hydrogen. In certain embodiments, R₃ is alkylor substituted alkyl. In certain embodiments, R₃ is alkenyl orsubstituted alkenyl. In certain embodiments, R₃ is alkynyl orsubstituted alkynyl. In certain embodiments, R₃ is alkoxy or substitutedalkoxy. In certain embodiments, R₃ is amino or substituted amino. Incertain embodiments, R₃ is carboxyl or carboxyl ester. In certainembodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃ is acylamino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R₃ is alkyl or substituted alkyl. For example,R₃ may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₃ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₃ ismethyl.

In certain embodiments, R₂ and R₃ are each independently selected fromalkyl and substituted alkyl. For example, R₂ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R₃ may be alkyl or substitutedalkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R₂ and R₃ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R₂and R₃ are each methyl.

In certain embodiments, X₁ is selected from C, N, O and S. In certainembodiments, X₁ is C. In certain embodiments, X₁ is N. In certainembodiments, X₁ is O. In certain embodiments, X₁ is S.

In certain embodiments, X₂ is selected from C, N, O and S. In certainembodiments, X₂ is C. In certain embodiments, X₂ is N. In certainembodiments, X₂ is O. In certain embodiments, X₂ is S.

In certain embodiments, X₃ is selected from C, N, O and S. In certainembodiments, X₃ is C. In certain embodiments, X₃ is N. In certainembodiments, X₃ is O. In certain embodiments, X₃ is S.

In certain embodiments, X₄ is selected from C, N, O and S. In certainembodiments, X₄ is C. In certain embodiments, X₄ is N. In certainembodiments, X₄ is O. In certain embodiments, X₄ is S.

Various combinations of X₁, X₂, X₃ and X₄ are possible. For example, incertain embodiments, each of X₁, X₂, X₃ and X₄ is C. In other instances,three of X₁, X₂, X₃ and X₄ are C and one of X₁, X₂, X₃ and X₄ is N. Inother embodiments, two of X₁, X₂, X₃ and X₄ are C and two of X₁, X₂, X₃and X₄ are N. In other embodiments, one of X₁, X₂, X₃ and X₄ is C andthree of X₁, X₂, X₃ and X₄ is are N. Other combinations of C, N, O and Sare possible for X₁, X₂, X₃ and X₄ as desired.

In certain embodiments, Y₁ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₁ is hydrogen. In certain embodiments, Y₁ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₁ is F. Incertain embodiments, Y₁ is Cl. In certain embodiments, Y₁ is Br. Incertain embodiments, Y₁ is I. In certain embodiments, Y₁ is alkyl orsubstituted alkyl. In certain embodiments, Y₁ is alkenyl or substitutedalkenyl. In certain embodiments, Y₁ is alkynyl or substituted alkynyl.In certain embodiments, Y₁ is alkoxy or substituted alkoxy. In certainembodiments, Y₁ is amino or substituted amino. In certain embodiments,Y₁ is carboxyl or carboxyl ester. In certain embodiments, Y₁ is acyl oracyloxy. In certain embodiments, Y₁ is acyl amino or amino acyl. Incertain embodiments, Y₁ is alkylamide or substituted alkylamide. Incertain embodiments, Y₁ is sulfonyl. In certain embodiments, Y₁ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₁ is arylor substituted aryl. In certain embodiments, Y₁ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₁ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₁ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₂ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₂ is hydrogen. In certain embodiments, Y₂ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₂ is F. Incertain embodiments, Y₂ is Cl. In certain embodiments, Y₂ is Br. Incertain embodiments, Y₂ is I. In certain embodiments, Y₂ is alkyl orsubstituted alkyl. In certain embodiments, Y₂ is alkenyl or substitutedalkenyl. In certain embodiments, Y₂ is alkynyl or substituted alkynyl.In certain embodiments, Y₂ is alkoxy or substituted alkoxy. In certainembodiments, Y₂ is amino or substituted amino. In certain embodiments,Y₂ is carboxyl or carboxyl ester. In certain embodiments, Y₂ is acyl oracyloxy. In certain embodiments, Y₂ is acyl amino or amino acyl. Incertain embodiments, Y₂ is alkylamide or substituted alkylamide. Incertain embodiments, Y₂ is sulfonyl. In certain embodiments, Y₂ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₂ is arylor substituted aryl. In certain embodiments, Y₂ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₂ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₂ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₃ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₃ is hydrogen. In certain embodiments, Y₃ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₃ is F. Incertain embodiments, Y₃ is Cl. In certain embodiments, Y₃ is Br. Incertain embodiments, Y₃ is I. In certain embodiments, Y₃ is alkyl orsubstituted alkyl. In certain embodiments, Y₃ is alkenyl or substitutedalkenyl. In certain embodiments, Y₃ is alkynyl or substituted alkynyl.In certain embodiments, Y₃ is alkoxy or substituted alkoxy. In certainembodiments, Y₃ is amino or substituted amino. In certain embodiments,Y₃ is carboxyl or carboxyl ester. In certain embodiments, Y₃ is acyl oracyloxy. In certain embodiments, Y₃ is acyl amino or amino acyl. Incertain embodiments, Y₃ is alkylamide or substituted alkylamide. Incertain embodiments, Y₃ is sulfonyl. In certain embodiments, Y₃ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₃ is arylor substituted aryl. In certain embodiments, Y₃ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₃ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₃ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, Y₄ is selected from hydrogen, halogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, Y₄ is hydrogen. In certain embodiments, Y₄ ishalogen, such as F, Cl, Br or I. In certain embodiments, Y₄ is F. Incertain embodiments, Y₄ is Cl. In certain embodiments, Y₄ is Br. Incertain embodiments, Y₄ is I. In certain embodiments, Y₄ is alkyl orsubstituted alkyl. In certain embodiments, Y₄ is alkenyl or substitutedalkenyl. In certain embodiments, Y₄ is alkynyl or substituted alkynyl.In certain embodiments, Y₄ is alkoxy or substituted alkoxy. In certainembodiments, Y₄ is amino or substituted amino. In certain embodiments,Y₄ is carboxyl or carboxyl ester. In certain embodiments, Y₄ is acyl oracyloxy. In certain embodiments, Y₄ is acyl amino or amino acyl. Incertain embodiments, Y₄ is alkylamide or substituted alkylamide. Incertain embodiments, Y₄ is sulfonyl. In certain embodiments, Y₄ isthioalkoxy or substituted thioalkoxy. In certain embodiments, Y₄ is arylor substituted aryl. In certain embodiments, Y₄ is heteroaryl orsubstituted heteroaryl. In certain embodiments, Y₄ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, Y₄ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, L is an optional linker. In certain embodiments,L is not present, and thus the nitrogen of the indole ring is directlybonded to W₁. In certain embodiments, L is present, and thus thenitrogen of the indole ring is indirectly bonded to W₁ through thelinker L.

In certain embodiments, L includes a group selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, L includes an alkyl or substituted alkyl group. Incertain embodiments, L includes an alkenyl or substituted alkenyl group.In certain embodiments, L includes an alkynyl or substituted alkynylgroup. In certain embodiments, L includes an alkoxy or substitutedalkoxy group. In certain embodiments, L includes an amino or substitutedamino group. In certain embodiments, L includes a carboxyl or carboxylester group. In certain embodiments, L includes an acyl amino group. Incertain embodiments, L includes an alkylamide or substituted alkylamidegroup. In certain embodiments, L includes an aryl or substituted arylgroup. In certain embodiments, L includes a heteroaryl or substitutedheteroaryl group. In certain embodiments, L includes a cycloalkyl orsubstituted cycloalkyl group. In certain embodiments, L includes aheterocyclyl or substituted heterocyclyl group.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

In certain embodiments, W₁ is selected from a drug, a detectable labeland a polypeptide. In certain embodiments, W₁ is a drug. In certainembodiments, W₁ is a detectable label. In certain embodiments, W₁ is apolypeptide.

Embodiments of the compound include a compound of formula (VIa):

In certain embodiments, the substituents in formula (VIa) are asdescribed above for formula (VI). For example, in certain embodiments,R₂ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments, R₂is hydrogen. In certain embodiments, R₂ is alkyl or substituted alkyl.In certain embodiments, R₂ is alkenyl or substituted alkenyl. In certainembodiments, R₂ is alkynyl or substituted alkynyl. In certainembodiments, R₂ is alkoxy or substituted alkoxy. In certain embodiments,R₂ is amino or substituted amino. In certain embodiments, R₂ is carboxylor carboxyl ester. In certain embodiments, R₂ is acyl or acyloxy. Incertain embodiments, R₂ is acyl amino or amino acyl. In certainembodiments, R₂ is alkylamide or substituted alkylamide. In certainembodiments, R₂ is sulfonyl. In certain embodiments, R₂ is thioalkoxy orsubstituted thioalkoxy. In certain embodiments, R₂ is aryl orsubstituted aryl. In certain embodiments, R₂ is heteroaryl orsubstituted heteroaryl. In certain embodiments, R₂ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, R₂ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments of formula (VIa), R₂ is alkyl or substitutedalkyl. For example, R₂ may be alkyl or substituted alkyl, such as,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl). In some cases, R₂ is methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₂ is methyl.

In certain embodiments of formula (VIa), R₃ is selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, R₃ is hydrogen. In certainembodiments, R₃ is alkyl or substituted alkyl. In certain embodiments,R₃ is alkenyl or substituted alkenyl. In certain embodiments, R₃ isalkynyl or substituted alkynyl. In certain embodiments, R₃ is alkoxy orsubstituted alkoxy. In certain embodiments, R₃ is amino or substitutedamino. In certain embodiments, R₃ is carboxyl or carboxyl ester. Incertain embodiments, R₃ is acyl or acyloxy. In certain embodiments, R₃is acyl amino or amino acyl. In certain embodiments, R₃ is alkylamide orsubstituted alkylamide. In certain embodiments, R₃ is sulfonyl. Incertain embodiments, R₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R₃ is aryl or substituted aryl. In certainembodiments, R₃ is heteroaryl or substituted heteroaryl. In certainembodiments, R₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (VIa), R₃ is alkyl or substitutedalkyl. For example, R₃ may be alkyl or substituted alkyl, such as,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆substituted alkyl). In some cases, R₃ is methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, or the like. Incertain cases, R₃ is methyl.

In certain embodiments of formula (VIa), R₂ and R₃ are eachindependently selected from alkyl and substituted alkyl. For example, R₂may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl), and R₃may be alkyl or substituted alkyl, such as, C₁-C₁₀ alkyl or C₁-C₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R₂ and R₃ are each independently selected from methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, t-butyl, orthe like. In certain cases, R₂ and R₃ are each methyl.

In certain embodiments of formula (VIa), Y₁ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₁ is hydrogen. Incertain embodiments, Y₁ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₁ is F. In certain embodiments, Y₁ is Cl. In certainembodiments, Y₁ is Br. In certain embodiments, Y₁ is I. In certainembodiments, Y₁ is alkyl or substituted alkyl. In certain embodiments,Y₁ is alkenyl or substituted alkenyl. In certain embodiments, Y₁ isalkynyl or substituted alkynyl. In certain embodiments, Y₁ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₁ is amino or substitutedamino. In certain embodiments, Y₁ is carboxyl or carboxyl ester. Incertain embodiments, Y₁ is acyl or acyloxy. In certain embodiments, Y₁is acyl amino or amino acyl. In certain embodiments, Y₁ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₁ is sulfonyl. Incertain embodiments, Y₁ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₁ is aryl or substituted aryl. In certainembodiments, Y₁ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₁ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₁ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (VIa), Y₂ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₂ is hydrogen. Incertain embodiments, Y₂ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₂ is F. In certain embodiments, Y₂ is Cl. In certainembodiments, Y₂ is Br. In certain embodiments, Y₂ is I. In certainembodiments, Y₂ is alkyl or substituted alkyl. In certain embodiments,Y₂ is alkenyl or substituted alkenyl. In certain embodiments, Y₂ isalkynyl or substituted alkynyl. In certain embodiments, Y₂ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₂ is amino or substitutedamino. In certain embodiments, Y₂ is carboxyl or carboxyl ester. Incertain embodiments, Y₂ is acyl or acyloxy. In certain embodiments, Y₂is acyl amino or amino acyl. In certain embodiments, Y₂ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₂ is sulfonyl. Incertain embodiments, Y₂ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₂ is aryl or substituted aryl. In certainembodiments, Y₂ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₂ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₂ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (VIa), Y₃ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₃ is hydrogen. Incertain embodiments, Y₃ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₃ is F. In certain embodiments, Y₃ is Cl. In certainembodiments, Y₃ is Br. In certain embodiments, Y₃ is I. In certainembodiments, Y₃ is alkyl or substituted alkyl. In certain embodiments,Y₃ is alkenyl or substituted alkenyl. In certain embodiments, Y₃ isalkynyl or substituted alkynyl. In certain embodiments, Y₃ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₃ is amino or substitutedamino. In certain embodiments, Y₃ is carboxyl or carboxyl ester. Incertain embodiments, Y₃ is acyl or acyloxy. In certain embodiments, Y₃is acyl amino or amino acyl. In certain embodiments, Y₃ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₃ is sulfonyl. Incertain embodiments, Y₃ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₃ is aryl or substituted aryl. In certainembodiments, Y₃ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₃ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₃ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (VIa), Y₄ is selected from hydrogen,halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, Y₄ is hydrogen. Incertain embodiments, Y₄ is halogen, such as F, Cl, Br or I. In certainembodiments, Y₄ is F. In certain embodiments, Y₄ is Cl. In certainembodiments, Y₄ is Br. In certain embodiments, Y₄ is I. In certainembodiments, Y₄ is alkyl or substituted alkyl. In certain embodiments,Y₄ is alkenyl or substituted alkenyl. In certain embodiments, Y₄ isalkynyl or substituted alkynyl. In certain embodiments, Y₄ is alkoxy orsubstituted alkoxy. In certain embodiments, Y₄ is amino or substitutedamino. In certain embodiments, Y₄ is carboxyl or carboxyl ester. Incertain embodiments, Y₄ is acyl or acyloxy. In certain embodiments, Y₄is acyl amino or amino acyl. In certain embodiments, Y₄ is alkylamide orsubstituted alkylamide. In certain embodiments, Y₄ is sulfonyl. Incertain embodiments, Y₄ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, Y₄ is aryl or substituted aryl. In certainembodiments, Y₄ is heteroaryl or substituted heteroaryl. In certainembodiments, Y₄ is cycloalkyl or substituted cycloalkyl. In certainembodiments, Y₄ is heterocyclyl or substituted heterocyclyl.

In certain embodiments of formula (VIa), L is an optional linker. Incertain embodiments, L is not present, and thus the nitrogen of theindole ring is directly bonded to W₁. In certain embodiments, L ispresent, and thus the nitrogen of the indole ring is indirectly bondedto W₁ through the linker L.

In certain embodiments of formula (VIa), L includes a group selectedfrom alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments of formula (VIa), L includes a polymer. Forexample, the polymer may include a polyalkylene glycol and derivativesthereof, including polyethylene glycol, methoxypolyethylene glycol,polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol (e.g., where thehomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers,polyvinylpyrrolidone, combinations thereof, and the like. In certainembodiments, the polymer is a polyalkylene glycol. In certainembodiments, the polymer is a polyethylene glycol.

In certain embodiments of formula (VIa), W₁ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₁ is adrug. In certain embodiments, W₁ is a detectable label. In certainembodiments, W₁ is a polypeptide.

Embodiments of the compound include a compound of formula (VIb):

In certain embodiments, the substituents in formula (VIb) are asdescribed above for formula (VI). For example, in certain embodiments, Lis an optional linker. In certain embodiments, L is not present, andthus the nitrogen of the indole ring is directly bonded to W₁. Incertain embodiments, L is present, and thus the nitrogen of the indolering is indirectly bonded to W₁ through the linker L.

In certain embodiments of formula (VIb), L includes a group selectedfrom alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments of formula (VIb), L includes a polymer. Forexample, the polymer may include a polyalkylene glycol and derivativesthereof, including polyethylene glycol, methoxypolyethylene glycol,polyethylene glycol homopolymers, polypropylene glycol homopolymers,copolymers of ethylene glycol with propylene glycol (e.g., where thehomopolymers and copolymers are unsubstituted or substituted at one endwith an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers,polyvinylpyrrolidone, combinations thereof, and the like. In certainembodiments, the polymer is a polyalkylene glycol. In certainembodiments, the polymer is a polyethylene glycol.

In certain embodiments of formula (VIb), W₁ is selected from a drug, adetectable label and a polypeptide. In certain embodiments, W₁ is adrug. In certain embodiments, W₁ is a detectable label. In certainembodiments, W₁ is a polypeptide.

Target Polypeptides

Any of a wide variety of polypeptides can be modified to be conjugatedto a second moiety as described above. Polypeptides suitable formodification include both proteins having a naturally-occurring aminoacid sequence, fragments of naturally-occurring polypeptides, andnon-naturally occurring polypeptides and fragments thereof.

The following are examples of classes and types of polypeptides whichare of interest for modification using the compounds and methodsdescribed herein to produce the polypeptide conjugates described herein.

Therapeutic Polypeptides

In certain embodiments, the methods of producing a conjugate are appliedto modification of polypeptides that may provide for a therapeuticbenefit, such as those polypeptides for which attachment to a moiety canprovide for one or more of, for example, an increase in serum half-life,a decrease in an adverse immune response, additional or alternatebiological activity or functionality, and the like, or other benefit orreduction of an adverse side effect. Where the therapeutic polypeptideis an antigen for a vaccine, modification can provide for an enhancedimmunogenicity of the polypeptide.

Examples of classes of therapeutic proteins include those that arecytokines, chemokines, growth factors, hormones, antibodies, andantigens. Further examples include, but are not limited to, thefollowing: erythropoietin (EPO, e.g., native EPO or synthetic EPO (see,e.g., US 2003/0191291), such as, but not limited to, e.g., PROCRIT®,EPREX®, or EPOGEN® (epoetin-α), ARANESP® (darbepoietin-α), NEORECORMON®,EPOGIN® (epoetin-β), and the like); a growth hormone (e.g., asomatotropin, e.g., GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®,SEROSTIM®, HUMATROPE®, etc.); human growth hormone (hGH); bovine growthhormone (bGH); follicle stimulating hormone (FSH); interferon (e.g.,IFN-7, IFN-α, IFN-β, IFN-ω; IFN-τ, consensus interferon, and the like);insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.),insulin-like growth factor (e.g., IGF-I, IGF-II); blood factors (e.g.,Factor X, tissue plasminogen activator (TPA), and the like, such as, butnot limited to, e.g., ACTIVASE® (alteplase) tissue plasminogenactivator, NOVOSEVEN® (recombinant human factor VIIa), Factor VIIa,Factor VIII (e.g., KOGENATE®), Factor IX, 0-globin, hemoglobin, and thelike); colony stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g.,NEUPOGEN® (filgrastim)), macrophage-CSF (M-CSF),granulocyte-macrophage-CSF (GM-CSF), Neulasta (pegfilgrastim),granulocyte-monocyte colony stimulating factor, megakaryocyte colonystimulating factor, and the like), transforming growth factors (e.g.,TGF-beta, TGF-alpha); interleukins (e.g., IL-1, IL-2 (e.g., Proleukin®),IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, and the like); a growthfactor (e.g., epidermal growth factor (EGF), platelet-derived growthfactor (PDGF, e.g., REGRANEX® (beclapermin)), fibroblast growth factors(FGFs, e.g., aFGF, bFGF, such as FIBLAST® (trafermin)), glial cellline-derived growth factor (GDNF), nerve growth factor (NGF), stem cellfactor (e.g., STEMGEN® (ancestim)), keratinocyte growth factor, ahepatocyte growth factor, and the like); a soluble receptor (e.g., aTNF-α-binding soluble receptor such as ENBREL® (etanercept), a solubleVEGF receptor, a soluble interleukin receptor, a soluble γ/δ T cellreceptor, and the like); an enzyme (e.g., α-glucosidase, CERAZYME®(imiglucarase, β-glucocerebrosidase, CEREDASE® (alglucerase); an enzymeactivator (e.g., tissue plasminogen activator); a chemokine (e.g.,IP-10, Mig, Groα/IL-8, regulated and normal T cell expressed andsecreted (RANTES), MIP-1α, MIP-1β, MCP-1, PF-4, and the like); anangiogenic agent (e.g., vascular endothelial growth factor (VEGF); ananti-angiogenic agent (e.g., a soluble VEGF receptor); a proteinvaccine; a neuroactive peptide such as bradykinin, cholecystokinin,gastin, secretin, oxytocin, gonadotropin-releasing hormone,beta-endorphin, enkephalin, substance P, somatostatin, galanin, growthhormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin,motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin,insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasinghormone, vasoactive intestinal peptide, a sleep peptide, etc.; otherproteins such as a thrombolytic agent, an atrial natriuretic peptide,bone morphogenic protein, thrombopoietin, relaxin, glial fibrillaryacidic protein, follicle stimulating hormone, a human alpha-1antitrypsin, a leukemia inhibitory factor, a transforming growth factor,a tissue factor, an insulin-like growth factor, a luteinizing hormone, afollicle stimulating hormone, a macrophage activating factor, tumornecrosis factor, a neutrophil chemotactic factor, a nerve growth factor,a tissue inhibitor of metalloproteinases; a vasoactive intestinalpeptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitoryfactor; an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); and thelike. It will be readily appreciated that native forms of the abovetherapeutic proteins are also of interest as target polypeptides in thepresent disclosure.

Further examples include antibodies, e.g., polyclonal antibodies,monoclonal antibodies, humanized antibodies, antigen-binding fragments(e.g., F(ab)′, Fab, Fv), single chain antibodies, and the like (e.g.,RITUXAN® (rituximab); REMICADE® (infliximab); HERCEPTIN® (trastuzumab);HUMIRA™ (adalimumab); XOLAIR® (omalizumab); BEXXAR® (tositumomab);RAPTIVA™ (efalizumab); ERBITUX™ (cetuximab); and the like). In someinstances, antibodies include antibodies that specifically bind to atumor antigen, an immune cell antigen (e.g., CD4, CD8, and the like), anantigen of a microorganism, particularly a pathogenic microorganism(e.g., a bacterial, viral, fungal, or parasitic antigen), and the like.

In some instances, the methods, conjugates and compounds describedherein can be applied to provide for a moiety (e.g., a water-solublepolymer) at a native or engineered site of glycosylation, such as foundin hyperglycosylated forms of a therapeutic protein.

The biological activity of a modified target polypeptide can be assayedaccording to methods known in the art. Modified polypeptides that retainat least one desired pharmacologic activity of the corresponding parentprotein are of interest.

Immunogenic Compositions

The methods, conjugates and compounds disclosed herein also findapplication in production of components of immunogenic compositions(e.g., therapeutic vaccines). For example, the compounds can be used tofacilitate attachment of moieties that increase serum half-life of apolypeptide antigen, that increase immunogenicity of the polypeptide, orthat link a non-amino acid antigen to a polypeptide carrier. In thisregard, the compounds can be used to facilitate modification ofmicrobial antigens (e.g., a bacterial, viral, fungal, or parasiticantigen), tumor antigens, and other antigens which are of interest foradministration to a subject to elicit an immune response in the subject.Also of interest is modification of antigens that are useful ineliciting antibodies which can be useful as research tools.

Further examples of polypeptides of interest for modification using thecompounds disclosed herein include those that are of interest fordetection or functional monitoring in an assay (e.g., as a researchtool, in a drug screening assay, and the like). Examples of polypeptidesof this type include receptors (e.g., G-protein coupled receptors(GPCRs, including orphan GPCRs)), receptor ligands (includingnaturally-occurring and synthetic), protein channels (e.g., ion channels(e.g., potassium channels, calcium channels, sodium channels, and thelike), and other polypeptides. In some embodiments, modification of cellsurface-associated polypeptides, such as transmembrane polypeptides) isof interest, for example where such modification is accomplished whilethe polypeptide is present in a membrane. Methods for modification of apolypeptide under physiological conditions are described further below.

Methods of Polypeptide Production

In general, the polypeptides described herein may be expressed inprokaryotes or eukaryotes in accordance with conventional ways,depending upon the purpose for expression. Thus, the present inventionfurther provides a host cell, e.g., a genetically modified host cellthat comprises a nucleic acid encoding a polypeptide.

Host cells for production (including large scale production) of anunconjugated or modified polypeptide suitable to form a conjugate asdescribed herein can be selected from any of a variety of available hostcells. Examples of host cells include those of a prokaryotic oreukaryotic unicellular organism, such as bacteria (e.g., Escherichiacoli strains, Bacillus spp. (e.g., B. subtilis), and the like) yeast orfungi (e.g., S. cerevisiae, Pichia spp., and the like), and other suchhost cells can be used. Examples of host cells originally derived from ahigher organism such as insects, vertebrates, including mammals, (e.g.,CHO, HEK, and the like), may be used as the expression host cells.

Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618 and CRL9096), CHO DG44 cells (Urlaub(1983) Cell 33:405), CHO-K1 cells (ATCC CCL-61), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems.

The expressed polypeptide can be recovered by any appropriate meansknown in the art. Further, any convenient protein purificationprocedures may be employed, where suitable protein purificationmethodologies are described in Guide to Protein Purification, (Deuthsered.) (Academic Press, 1990). For example, a lysate may prepared from acell comprising the expression vector expressing the desiredpolypeptide, and purified using high performance liquid chromatography(HPLC), exclusion chromatography, gel electrophoresis, affinitychromatography, and the like.

Methods for Modification of a Polypeptide

In certain embodiments, the polypeptide may be conjugated to a moiety ofinterest without first modifying the polypeptide. For instance, thepolypeptide may include one or more reactive groups suitable forconjugation to the moiety of interest (e.g., a moiety comprising acoupling moiety, such as a hydrazinyl-indole compound orhydrazinyl-indole derivative as described herein). In other embodiments,the polypeptide may be modified before conjugation to the moiety ofinterest. Modification of the polypeptide may produce a modifiedpolypeptide that contains one or more reactive groups suitable forconjugation to the moiety of interest.

In some cases, the polypeptide may be modified at one or more amino acidresidues to provide one or more reactive groups suitable for conjugationto the moiety of interest (e.g., a moiety comprising a coupling moiety,such as a hydrazinyl-indole compound or hydrazinyl-indole derivative asdescribed herein). For example, carbonyls introduced into a polypeptidecan be selectively reacted with α-nucleophiles, such as aminooxy- andhydrazide-bearing compounds. Chemistries selective for carbonylfunctional groups on a protein with enhanced kinetics, site selectivityand conjugate stability may result in improved bioconjugates and provideaccess to new products and therapeutic targets as disclosed herein.

In certain embodiments, the polypeptide may be modified to include analdehyde tag. By “aldehyde tag” or “ald-tag” is meant an amino acidsequence that contains an amino acid sequence derived from a sulfatasemotif which is capable of being converted, or which has been converted,by action of a formylglycine generating enzyme (FGE) to contain a2-formylglycine residue (referred to herein as “fGly”). The fGly residuegenerated by an FGE is often referred to in the literature as a“formylglycine”. Stated differently, the term “aldehyde tag” is usedherein to refer to an amino acid sequence comprising an “unconverted”sulfatase motif (i.e., a sulfatase motif in which the cysteine or serineresidues has not been converted to fGly by an FGE, but is capable ofbeing converted, such as a sulfatase motif with the sequence:L(C/S)TPSR) as well as to an amino acid sequence comprising a“converted” sulfatase motif (i.e., a sulfatase motif in which thecysteine or the serine residue has been converted to fGly by action ofan FGE, e.g., L(fGly)TPSR). By “conversion” as used in the context ofaction of a formylglycine generating enzyme (FGE) on a sulfatase motifrefers to biochemical modification of a cysteine or serine residue in asulfatase motif to a formylglycine (fGly) residue (e.g., Cys to fGly, orSer to fGly). Additional aspects of aldehyde tags and uses thereof insite-specific protein modification are described in U.S. Pat. No.7,985,783, the disclosure of which is incorporated herein by reference.

Conversion of a polypeptide to include fGly can be accomplished bycell-based (in vivo) or cell-free methods (in vitro). Similarly,modification of a polypeptide to produce a polypeptide suitable forconjugation (e.g., modification to produce a polypeptide containing areactive group suitable for conjugation) can be accomplished bycell-based (in vivo) or cell-free methods (in vitro).

Alternatively, isolated, unmodified polypeptides can be isolatedfollowing recombinant production in a host cell lacking a suitableenzyme or by synthetic production. The isolated polypeptide may then becontacted with a suitable enzyme under conditions to provide for thedesired modification of the polypeptide to include fGly. The polypeptidecan be unfolded by methods known in the art (e.g., using heat,adjustment of pH, chaotropic agents, (e.g., urea, and the like), organicsolvents (e.g., hydrocarbons: octane, benzene, chloroform), etc.) andthe denatured protein contacted with a suitable enzyme. The modifiedpolypeptide can then be refolded under suitable conditions.

In some cases, the modified polypeptide containing the fGly residue maybe conjugated to the moiety of interest by reaction of the fGly with acompound as described herein (e.g., a compound containing a couplingmoiety, such as a hydrazinyl-indole or hydrazinyl-indole derivative asdescribed herein). For example, an fGly-containing polypeptide may beisolated from a production source (e.g., recombinant host cellproduction, synthetic production), and contacted with a reactivepartner-containing drug or other moiety (e.g., detectable label) underconditions suitable to provide for conjugation of the drug or othermoiety to the polypeptide. For example, the reactive partner-containingdrug or other moiety may include a reactive moiety (e.g., ahydrazinyl-indole compound or hydrazinyl-indole derivative as describedherein). The hydrazinyl-indole-containing drug or other moiety may bereacted with the polypeptide to produce a polypeptide conjugate asdescribed herein. For example, FIG. 1 , panels A and B, show schematicdrawings of polypeptide conjugates according to embodiments of thepresent disclosure. FIG. 1 , panel A, shows a reaction scheme for theproduction of a polypeptide conjugate that includes a hydrazinyl-indolecoupling moiety. In FIG. 1 , panel A, a polypeptide (indicated by theshaded sphere in the figure) that includes an fGly is reacted with ahydrazinyl-indole coupling moiety (e.g., a hydrazinyl-indole compound orhydrazinyl-indole derivative) to produce a polypeptide conjugate thatincludes the coupling moiety. As shown in FIG. 1 , panel A, thehydrazine of the hydrazinyl-indole coupling moiety undergoes anintramolecular cyclization to form a partially unsaturated pyrazole orpyridazine ring depending on whether n is 0 or 1, respectively. In FIG.1 , panel A, the shaded sphere may represent a polypeptide and R₄ may bea drug or a detectable label, however in other embodiments, the shadedsphere may represent a drug or a detectable label and R₄ may be apolypeptide. FIG. 1 , panel B, shows a reaction scheme for theproduction of a polypeptide conjugate that includes a differenthydrazinyl-indole coupling moiety. In FIG. 1 , panel B, a polypeptidethat includes an fGly is reacted with the hydrazinyl-indole couplingmoiety to produce a polypeptide conjugate that includes the couplingmoiety. As shown in FIG. 1 , panel B, the hydrazine of thehydrazinyl-indole coupling moiety undergoes an intramolecularcyclization to form a partially unsaturated pyridazine or 1,2-diazepinering depending on whether n is 0 or 1, respectively. In FIG. 1 , panelB, the shaded sphere may represent a polypeptide and R₄ may be a drug ora detectable label, however in other embodiments, the shaded sphere mayrepresent a drug or a detectable label and R₄ may be a polypeptide.

Polypeptide Conjugates

The polypeptides can be subjected to conjugation to provide forattachment of a wide variety of moieties. Examples of moieties ofinterest include, but are not limited to, a drug, a detectable label, asmall molecule, a water-soluble polymer, a peptide, and the like. Thus,the present disclosure provides a polypeptide conjugate as describedabove.

The moiety of interest is provided as a component of a reactive partnerfor reaction with a residue of a polypeptide. In certain embodiments,the methods of polypeptide conjugation are compatible with reactionconditions suitable for the polypeptide. For example, the reactionconditions may include a reaction mixture that includes water. In somecases, the reaction mixture may have a pH compatible with thepolypeptide, such as, but not limited to, a pH of 4 to 11, or a pH of 5to 10, or a pH of 6 to 9, or a pH of 6 to 8. In certain instances, thereaction mixture has a pH of 7. In some embodiments, the reactionconditions are performed at a temperature compatible with thepolypeptide. For example, the reaction conditions may be at atemperature of 20° C. to 45° C., such as 25° C. to 40° C., or 30° C. to40° C., or 35° C. to 40° C. In some cases, the reaction conditions areat room temperature (e.g., 25° C.). In some instances, the reactionconditions are at a temperature of 37° C.

Provided the present disclosure, the ordinarily skilled artisan canreadily adapt any of a variety of moieties to provide a reactive partnerfor conjugation to a polypeptide as contemplated herein. The ordinarilyskilled artisan will appreciate that factors such as pH and sterichindrance (i.e., the accessibility of the modified amino acid residue toreaction with a reactive partner of interest) are of importance.Modifying reaction conditions to provide for optimal conjugationconditions is well within the skill of the ordinary artisan, and isroutine in the art. Where conjugation is conducted with a polypeptidepresent in or on a living cell, the conditions are selected so as to bephysiologically compatible. For example, the pH can be droppedtemporarily for a time sufficient to allow for the reaction to occur butwithin a period tolerated by the cell (e.g., from about 30 min to 1hour). Physiological conditions for conducting modification ofpolypeptides on a cell surface can be similar to those used in aketone-azide reaction in modification of cells bearing cell-surfaceazides (see, e.g., U.S. Pat. No. 6,570,040).

In certain embodiments, the present disclosure provides a polypeptideconjugate, where the polypeptide is an antibody. As such, embodimentsinclude an antibody conjugated to a moiety of interest, where anantibody conjugated to a moiety of interest is referred to as an“antibody conjugate.” An Ig polypeptide generally includes at least anIg heavy chain constant region or an Ig light chain constant region, andcan further include an Ig variable region (e.g., a V_(L) region and/or aV_(H) region). Ig heavy chain constant regions include Ig constantregions of any heavy chain isotype, non-naturally occurring Ig heavychain constant regions (including consensus Ig heavy chain constantregions). An Ig constant region can be modified to be conjugated to amoiety of interest, where the moiety of interest is present in oradjacent a solvent-accessible loop region of the Ig constant region.

In some cases, an antibody conjugate of the present disclosure caninclude: 1) Ig heavy chain constant region conjugated to one or moremoieties of interest, and an Ig light chain constant region conjugatedto one or more moieties of interest; 2) an Ig heavy chain constantregion conjugated to one or more moieties of interest, and an Ig lightchain constant region that is not conjugated to a moiety of interest; or3) an Ig heavy chain constant region that is not conjugated to a moietyof interest, and an Ig light chain constant region conjugated to one ormore moieties of interest. A subject antibody conjugate can also includevariable VH and/or VL domains. As described above, the one or moremoieties of interest may be conjugated to the Ig heavy chain constantregion or the Ig light chain constant region at a single amino acidresidue (e.g., one or two moieties of interest conjugated to a singleamino acid residue), or conjugated to the Ig heavy chain constant regionand/or the Ig light chain constant region at two or more different aminoacid residues.

An antibody conjugate of the present disclosure can include, as theconjugated moiety, any of a variety of compounds, as described herein,e.g., a drug (e.g., a peptide drug, a small molecule drug, and thelike), a water-soluble polymer, a detectable label, a synthetic peptide,etc.

An antibody conjugate can have any of a variety of antigen-bindingspecificities, as described above, including, e.g., an antigen presenton a cancer cell; an antigen present on an autoimmune cell; an antigenpresent on a pathogenic microorganism; an antigen present on avirus-infected cell (e.g., a human immunodeficiency virus-infectedcell), e.g., CD4 or gp120; an antigen present on a diseased cell; andthe like. For example, an antibody conjugate can bind an antigen, asnoted above, where the antigen is present on the surface of the cell. Anantibody conjugate of the present disclosure can bind antigen with asuitable binding affinity, e.g., from 5×10⁻⁶ M to 10⁻⁷ M, from 10⁻⁷ M to5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸ M, from 10⁻⁸ M to 5×10⁻⁸ M, from 5×10⁻⁸M to 10⁻⁹ M, or a binding affinity greater than 10⁻⁹ M.

As non-limiting examples, a subject antibody conjugate can bind anantigen present on a cancer cell (e.g., a tumor-specific antigen; anantigen that is over-expressed on a cancer cell; etc.), and theconjugated moiety can be a drug (e.g., a small molecule drug, a peptidedrug, etc.). For example, a subject antibody conjugate can be specificfor CD19, where the conjugated moiety is a drug (e.g., a small moleculedrug, a peptide drug, etc.). As another example, a subject antibodyconjugate can be specific for CD22, where the conjugated moiety can be adrug (e.g., a small molecule drug, a peptide drug, etc.).

As further non-limiting examples, a subject antibody conjugate can bindan antigen present on a cell infected with a virus (e.g., where theantigen is encoded by the virus; where the antigen is expressed on acell type that is infected by a virus; etc.), and the conjugated moietycan be a viral fusion inhibitor. For example, a subject antibodyconjugate can bind CD4, and the conjugated moiety can be a viral fusioninhibitor. As another example, a subject antibody conjugate can bindgp120, and the conjugated moiety can be a viral fusion inhibitor.

Embodiments of the present disclosure also include polypeptideconjugates where the polypeptide is a carrier protein. For example,carrier proteins can be covalently and site-specifically bound to drugto provide a drug-containing scaffold. A carrier protein can besite-specifically conjugated to a covalently bound molecule of interest,such as a drug (e.g., a peptide, a small molecule drug, and the like),detectable label, etc. In certain embodiments, drug-scaffold conjugatescan provide for enhanced serum half-life of the drug.

In general a “carrier protein” is a protein that is biologically inert,is susceptible to modification as disclosed herein, and which canprovide for solvent-accessible presentation of the moiety of interestconjugated to the carrier protein through a modified amino acid residuein the carrier protein (e.g., through an oxime or hydrazone bond withinthe converted sulfatase motif of an aldehyde tagged carrier protein) ina physiological environment. “Biologically inert” is meant to indicatethe carrier protein exhibits clinically insignificant or no detectablebiological activity when administered to the appropriate subject, suchas when administered to a human subject. Thus, carrier proteins arebiologically inert in that they, for example, are of low immunogenicity,do not exhibit significant or detectable targeting properties (e.g., donot exhibit significant or detectable activity in binding to a specificreceptor), and exhibit little or no detectable biological activity thatmay interfere with activity of the moiety (e.g., drug or detectablelabel) conjugated to the aldehyde-tagged carrier protein. By “lowimmunogenicity” is meant that the carrier protein elicits little or nodetectable immune response upon administration to a subject, such as amammalian subject, e.g., a human subject. Carrier proteins can beprovided in monomeric or multimeric (e.g., dimeric) forms.

Carrier proteins having a three-dimensional structure when folded thatprovides for multiple different solvent-accessible sites that areamenable to modification (and thus conjugation to a moiety of interest)are of interest. In general, carrier proteins of interest are those thatare of a size and three-dimensional folded structure so as to providefor presentation of the conjugated moiety of interest on solventaccessible surfaces in a manner that is sufficiently spatially separatedso as to provide for activity and bioavailability of the conjugatedmoiety or moieties of interest. The carrier protein may be selectedaccording to a variety of factors including, but not limited to, themoiety (e.g., drug or detectable label) to be conjugated to the carrierprotein.

Accordingly, any of a wide variety of polypeptides can be suitable foruse as carrier proteins for use in the carrier protein conjugates of thepresent disclosure. Such carrier proteins can include those having anaturally-occurring amino acid sequence, fragments ofnaturally-occurring polypeptides, and non-naturally occurringpolypeptides and fragments thereof.

Examples of carrier proteins include, but are not limited to, albuminand fragments thereof (e.g., human serum albumin, bovine serum albumin,and the like), transferrin and fragments thereof (e.g. humantransferrin), and Fc fragments having reduced binding to a mammalian Fcreceptor, particularly a human Fc receptor (e.g., a modified Fc fragmentof an antibody (e.g., IgG), such as a mammalian antibody, e.g., a humanantibody). Examples of modified Fc fragments having reduced Fc receptorbinding are exemplified by the Fc fragments of Herceptin (trastuzumab)and Rituxan (Rituximab), which contain point mutations that provide forreduced Fc receptor binding (see, e.g., Clynes et al., Nature Medicine(2000), 6, 443-446). Alternatively or in addition, the isotype of the Fcfragment can be selected according to a desired level of Fc receptorbinding (e.g., use of an Fc fragment of an IgG4 isotype human heavychain constant region rather than from IgG1 or IgG3. (see, e.g., FridmanFASEB J 1991 September; 5 (12): 2684-90). In general, carrier proteinscan be at least about 4 kDa (e.g., about 50 amino acid residues inlength), usually at least about 25 kDa, and can be larger in size (e.g.,transferrin has a molecular weight of 90 kDa while Fc fragments can havemolecular weights of 30 kDa to 50 kDa).

The conjugates described herein can be used for a variety ofapplications including, but not limited to, visualization usingfluorescence or epitope labeling (e.g., electron microscopy using goldparticles equipped with reactive groups for conjugation to the compoundsand conjugates described herein); protein immobilization (e.g., proteinmicroarray production); protein dynamics and localization studies andapplications; and conjugation of proteins with a moiety of interest(e.g., moieties that improve a parent protein's half-life (e.g.,poly(ethylene glycol)), targeting moieties (e.g., to enhance delivery toa site of action), and biologically active moieties (e.g., a therapeuticmoiety).

The polypeptide conjugate may include a polypeptide conjugated to amoiety or moieties that provide for one or more of a wide variety offunctions or features. In general, examples of moieties include, but arenot limited to, the following: detectable labels (e.g., fluorescentlabels or fluorophores); light-activated dynamic moieties (e.g.,azobenzene mediated pore closing, azobenzene mediated structuralchanges, photodecaging recognition motifs); water soluble polymers(e.g., PEGylation); purification tags (e.g., to facilitate isolation byaffinity chromatography (e.g., attachment of a FLAG epitope); membranelocalization domains (e.g., lipids or glycophosphatidylinositol(GPI)-type anchors); immobilization tags (e.g., to facilitate attachmentof the polypeptide to a surface, including selective attachment); drugs(e.g., to facilitate drug targeting, e.g., through attachment of thedrug to an antibody); targeted delivery moieties, (e.g., ligands forbinding to a target receptor (e.g., to facilitate viral attachment,attachment of a targeting protein present on a liposome, etc.)), and thelike.

Specific, non-limiting examples are provided below.

Drugs for Conjugation to a Polypeptide

Any of a number of drugs are suitable for use, or can be modified to berendered suitable for use, as a reactive partner to conjugate to apolypeptide. Examples of drugs include small molecule drugs and peptidedrugs. Thus, the present disclosure provides drug-polypeptideconjugates.

“Small molecule drug” as used herein refers to a compound, e.g., anorganic compound, which exhibits a pharmaceutical activity of interestand which is generally of a molecular weight of 800 Da or less, or 2000Da or less, but can encompass molecules of up to 5 kDa and can be aslarge as 10 kDa. A small inorganic molecule refers to a moleculecontaining no carbon atoms, while a small organic molecule refers to acompound containing at least one carbon atom.

“Peptide drug” as used herein refers to amino-acid containing polymericcompounds, and is meant to encompass naturally-occurring andnon-naturally-occurring peptides, oligopeptides, cyclic peptides,polypeptides, and proteins, as well as peptide mimetics. The peptidedrugs may be obtained by chemical synthesis or be produced from agenetically encoded source (e.g., recombinant source). Peptide drugs canrange in molecular weight, and can be from 200 Da to 10 kDa or greaterin molecular weight.

In some cases, the drug is a cancer chemotherapeutic agent. For example,where the polypeptide is an antibody (or fragment thereof) that hasspecificity for a tumor cell, the antibody can be modified as describedherein to include a modified amino acid, which can be subsequentlyconjugated to a cancer chemotherapeutic agent. Cancer chemotherapeuticagents include non-peptidic (i.e., non-proteinaceous) compounds thatreduce proliferation of cancer cells, and encompass cytotoxic agents andcytostatic agents. Non-limiting examples of chemotherapeutic agentsinclude alkylating agents, nitrosoureas, antimetabolites, antitumorantibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidiccompounds can also be used.

Suitable cancer chemotherapeutic agents include dolastatin and activeanalogs and derivatives thereof; and auristatin and active analogs andderivatives thereof. See, e.g., WO 96/33212, WO 96/14856, and U.S. Pat.No. 6,323,315. For example, dolastatin 10 or auristatin PE can beincluded in an antibody-drug conjugate of the present disclosure.Suitable cancer chemotherapeutic agents also include maytansinoids andactive analogs and derivatives thereof (see, e.g., EP 1391213; and Liuet al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); and duocarmycinsand active analogs and derivatives thereof (e.g., including thesynthetic analogues, KW-2189 and CB 1-TM1).

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel(Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®.Estrogens stimulate proliferation and differentiation; thereforecompounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other suitable chemotherapeutic agents include metal complexes, e.g.cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; andhydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomeraseinhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

Taxanes are suitable for use. “Taxanes” include paclitaxel, as well asany active taxane derivative or pro-drug. “Paclitaxel” (which should beunderstood herein to include analogues, formulations, and derivativessuch as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation ofdocetaxel), 10-desacetyl analogs of paclitaxel and3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs of paclitaxel) may bereadily prepared utilizing techniques known to those skilled in the art(see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949;5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtainedfrom a variety of commercial sources, including for example, SigmaChemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Biological response modifiers suitable for use include, but are notlimited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2)inhibitors of serine/threonine kinase activity; (3) tumor-associatedantigen antagonists, such as antibodies that bind specifically to atumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6)IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) inhibitors ofangiogenesis.

Methods for Modification of Drugs to Contain a Reactive Partner

Drugs to be conjugated to a polypeptide may be modified to incorporate areactive partner for reaction with the polypeptide. Where the drug is apeptide drug, the reactive moiety (e.g., aminooxy or hydrazide can bepositioned at an N-terminal region, the N-terminus, a C-terminal region,the C-terminus, or at a position internal to the peptide. For example,an example of a method involves synthesizing a peptide drug having anaminooxy group. In this example, the peptide is synthesized from aBoc-protected precursor. An amino group of a peptide can react with acompound comprising a carboxylic acid group and oxy-N-Boc group. As anexample, the amino group of the peptide reacts with3-(2,5-dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on thecompound comprising a carboxylic acid group and oxy-N-protecting groupcan include different number of carbons in the alkylene linker andsubstituents on the alkylene linker. The reaction between the aminogroup of the peptide and the compound comprising a carboxylic acid groupand oxy-N-protecting group occurs through standard peptide couplingchemistry. Examples of peptide coupling reagents that can be usedinclude, but not limited to, DCC (dicyclohexylcarbodiimide), DIC(diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP(1-benzotriazolediethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC(1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuricfluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidiniumhexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP(benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate), HBTU(O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate),TBTU (O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate), TSTU(O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate),HATU(N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1-ylmethylene]--N-methylmethanaminiumhexafluorophosphate N-oxide), BOP-Cl(bis(2-oxo-3-oxazolidinyl)phosphinic chloride), PyBOP((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidino)phosphoniumtetrafluorophopsphate), BrOP (bromotris(dimethylamino)phosphoniumhexafluorophosphate), DEPBT(3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP(bromotris(pyrrolidino)phosphonium hexafluorophosphate). As anon-limiting example, HOBt and DIC can be used as peptide couplingreagents.

Deprotection to expose the amino-oxy functionality is performed on thepeptide comprising an N-protecting group. Deprotection of theN-oxysuccinimide group, for example, occurs according to standarddeprotection conditions for a cyclic amide group. Deprotectingconditions can be found in Greene and Wuts, Protective Groups in OrganicChemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al.Certain deprotection conditions include a hydrazine reagent, aminoreagent, or sodium borohydride. Deprotection of a Boc protecting groupcan occur with TFA. Other reagents for deprotection include, but are notlimited to, hydrazine, methylhydrazine, phenylhydrazine, sodiumborohydride, and methylamine. The product and intermediates can bepurified by conventional means, such as HPLC purification.

The ordinarily skilled artisan will appreciate that factors such as pHand steric hindrance (i.e., the accessibility of the amino acid residueto reaction with a reactive partner of interest) are of importance,Modifying reaction conditions to provide for optimal conjugationconditions is well within the skill of the ordinary artisan, and isroutine in the art. Where conjugation is conducted with a polypeptidepresent in or on a living cell, the conditions are selected so as to bephysiologically compatible. For example, the pH can be droppedtemporarily for a time sufficient to allow for the reaction to occur butwithin a period tolerated by the cell (e.g., from about 30 min to 1hour). Physiological conditions for conducting modification ofpolypeptides on a cell surface can be similar to those used in aketone-azide reaction in modification of cells bearing cell-surfaceazides (see, e.g., U.S. Pat. No. 6,570,040).

Small molecule compounds containing, or modified to contain, anX-nucleophilic group that serves as a reactive partner with a compoundor conjugate disclosed herein are also contemplated for use as drugs inthe polypeptide-drug conjugates of the present disclosure. Generalmethods are known in the art for chemical synthetic schemes andconditions useful for synthesizing a compound of interest (see, e.g.,Smith and March, March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; orVogel, A Textbook of Practical Organic Chemistry, Including QualitativeOrganic Analysis, Fourth Edition, New York: Longman, 1978).

Peptide Drugs

In some cases, a conjugate comprises a covalently linked peptide.Suitable peptides include, but are not limited to, cytotoxic peptides;angiogenic peptides; anti-angiogenic peptides; peptides that activate Bcells; peptides that activate T cells; anti-viral peptides; peptidesthat inhibit viral fusion; peptides that increase production of one ormore lymphocyte populations; anti-microbial peptides; growth factors;growth hormone-releasing factors; vasoactive peptides; anti-inflammatorypeptides; peptides that regulate glucose metabolism; an anti-thromboticpeptide; an anti-nociceptive peptide; a vasodilator peptide; a plateletaggregation inhibitor; an analgesic; and the like.

In some embodiments, the peptide can be chemically synthesized toinclude a group reactive with an amino acid residue or a modified aminoacid residue of the polypeptide. A suitable synthetic peptide has alength of from 5 amino acids to 100 amino acids, or longer than 100amino acids; e.g., a suitable peptide has a length of from 5 amino acids(aa) to 10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, from 40 aa to 50 aa,from 50 aa to 60 aa, from 60 aa to 70 aa, from 70 aa to 80 aa, from 80aa to 90 aa, or from 90 aa to 100 aa.

In certain embodiments, a peptide can be modified to contain anα-nucleophile-containing moiety (e.g., an aminooxy or hydrazide moiety),e.g., can be reacted with an fGly-containing polypeptide to yield aconjugate in which the polypeptide and peptide are linked by a hydrazoneor oxime bond, respectively. Examples of methods of synthesizing apeptide, such that the synthetic peptide comprising a reactive groupreactive with an amino acid residue or a modified amino acid residue ofthe polypeptide, are described above.

Suitable peptides include, but are not limited to, hLF-11 (an 11-aminoacid N-terminal fragment of lactoferrin), an anti-microbial peptide;granulysin, an anti-microbial peptide; Plectasin (NZ2114; SAR 215500),an anti-microbial peptide; viral fusion inhibitors such as Fuzeon(enfuvirtide), TRI-1249 (T-1249; see, e.g., Matos et al. (2010) PLoS One5:e9830), TRI-2635 (T-2635; see, e.g., Eggink et al. (2009) J. Biol.Chem. 284:26941), T651, and TRI-1144; C5a receptor inhibitors such asPMX-53, JPE-1375, and JSM-7717; POT-4, a human complement factor C3inhibitor; Pancreate (an INGAP derivative sequence, a HIP-human proisletprotein); somatostatin; a somatostatin analog such as DEBIO 8609(Sanvar), octreotide, octreotide (C2L), octreotide QLT, octreotide LAR,Sandostatin LAR, SomaLAR, Somatuline (lanreotide), see, e.g., Deghenghiet al. (2001) Endocrine 14:29; TH9507 (Tesamorelin, a growthhormone-releasing factor); POL7080 (a protegrin analog, ananti-microbial peptide); relaxin; a corticotropin releasing factoragonist such as urotensin, sauvagine, and the like; a heat shock proteinderivative such as DiaPep277; a human immunodeficiency virus entryinhibitor; a heat shock protein-20 mimic such as AZX100; a thrombinreceptor activating peptide such as TP508 (Chrysalin); a urocortin 2mimic (e.g., a CRF2 agonist) such as urocortin-2; an immune activatorsuch as Zadaxin (thymalfasin; thymosin-α1), see, e.g., Sjogren (2004) J.Gastroenterol. Hepatol. 19:S69; a hepatitis C virus (HCV) entryinhibitorE2 peptide such as HCV3; an atrial natriuretic peptide such asHANP (Sun 4936; carperitide); an annexin peptide; a defensin(anti-microbial peptide) such as hBD2-4; a defensin (anti-microbialpeptide) such as hBD-3; a defensin (anti-microbial peptide) such asPMX-30063; a histatin (anti-microbial peptide) such as histatin-3,histatin-5, histatin-6, and histatin-9; a histatin (anti-microbialpeptide) such as PAC-113; an indolicidin (anti-microbial peptide) suchas MX-594AN (Omniganin; CLS001); an indolicidin (anti-microbial peptide)such as Omnigard (MBI-226; CPI-226); an anti-microbial peptide such asan insect cecropin; an anti-microbial peptide such as a lactoferrin(talactoferrin); an LL-37/cathelicidin derivative (an anti-microbialpeptide) such as P60.4 (OP-145); a magainin (an anti-microbial peptide)such as Pexiganan (MSI-78; Suponex); a protegrin (an anti-microbialpeptide) such as IB-367 (Iseganan); an agan peptide; a beta-natriureticpeptide such as Natrecor, or Noratak (Nesiritide), or ularitide;bivalarudin (Angiomax), a thrombin inhibitor; a C peptide derivative; acalcitonin such as Miacalcin (Fortical); an enkephalin derivative; anerythropoiesis-stimulating peptide such as Hematide; a gap junctionmodulator such as Danegaptide (ZP1609); a gastrin-releasing peptide; aghrelin; a glucagon-like peptide; a glucagon-like peptide-2 analog suchas ZP1846 or ZP1848; a glucosaminyl muramyl dipeptide such as GMDP; aglycopeptide antibiotic such as Oritavancin; a teicoplanin derivativesuch as Dalbavancin; a gonadotropin releasing hormone (GnRH) such asZoladex (Lupon) or Triptorelin; a histone deacetylase (HDAC) inhibitordepsipeptide such as PM02734 (Irvalec); an integrin such aseptifibatide; an insulin analog such as Humulog; a kahalalidedepsipeptide such as PM02734; a kallikrein inhibitor such as Kalbitor(ecallantide); an antibiotic such as Telavancin; a lipopeptide such asCubicin or MX-2401; a lutenizing hormone releasing hormone (LHRH) suchas goserelin; an LHRH synthetic decapeptide agonist analog such asTreistar (triptorelin pamoate); an LHRH such as Eligard; an M2 proteinchannel peptide inhibitor; metreleptin; a melanocortin receptor agonistpeptide such as bremalanotide/PT-141; a melanocortin; a muramyltripeptide such as Mepact (mifamurtide); a myelin basic protein peptidesuch as MBP 8298 (dirucotide); an N-type voltage-gated calcium channelblocker such as Ziconotide (Prialt); a parathyroid hormone peptide; aparathyroid analog such as 768974; a peptide hormone analog such asUGP281; a prostaglandin F2-α receptor inhibitor such as PDC31; aprotease inhibitor such as PPL-100; surfaxin; a thromobspondin-1 (TSP-1)mimetic such as CVX-045 or ABT 510; a vasoactive intestinal peptide;vasopressin; a Y2R agonist peptide such as RG7089; obinepeptide; andTM30339.

Detectable Labels

The conjugates, compounds and methods of the present disclosure can beused to conjugate a detectable label to polypeptide. Examples ofdetectable labels include, but are not limited to, fluorescent molecules(e.g., autofluorescent molecules, molecules that fluoresce upon contactwith a reagent, etc.), radioactive labels (e.g., ¹¹¹In, ¹²⁵I, ¹³¹I,²¹²B, ⁹⁰Y, ¹⁸⁶Rh and the like), biotin (e.g., to be detected throughreaction of biotin and avidin), fluorescent tags, imaging reagents, andthe like. Detectable labels also include peptides or polypeptides thatcan be detected by antibody binding, e.g., by binding of a detectablylabeled antibody or by detection of bound antibody through asandwich-type assay. Further examples of detectable labels include, butare not limited to, dye labels (e.g., chromophores, fluorophores, suchas, but not limited to, Alexa Fluor® fluorescent dyes (e.g., AlexaFluor® 350, 405, 430, 488, 532, 546, 555, 568, 594, 595, 610, 633, 635,647, 660, 680, 700, 750, 790, and the like)), biophysical probes (spinlabels, nuclear magnetic resonance (NMR) probes), Förster ResonanceEnergy Transfer (FRET)-type labels (e.g., at least one member of a FRETpair, including at least one member of a fluorophore/quencher pair),Bioluminescence Resonance Energy Transfer (BRET)-type labels (e.g., atleast one member of a BRET pair), immunodetectable tags (e.g., FLAG,His(6), and the like), localization tags (e.g., to identify associationof a tagged polypeptide at the tissue or molecular cell level (e.g.,association with a tissue type, or particular cell membrane), and thelike.

Attachment of Moieties for Delivery to a Target Site

Embodiments of the present disclosure also include a polypeptideconjugated to one or more moieties, such as, but not limited to, a drug(e.g., a small molecule drug), toxin, or other molecule for delivery toa target site (e.g., a cell) and which can provide for a pharmacologicalactivity or can serve as a target for delivery of other molecules.

Also contemplated are conjugates that include one of a pair of bindingpartners (e.g., a ligand, a ligand-binding portion of a receptor, areceptor-binding portion of a ligand, etc.). For example, the conjugatecan include a polypeptide that serves as a viral receptor and, uponbinding with a viral envelope protein or viral capsid protein,facilitates attachment of virus to the cell surface on which themodified polypeptide is expressed. Alternatively, the conjugate mayinclude an antigen that is specifically bound by an antibody (e.g.,monoclonal antibody), to facilitate detection and/or separation of hostcells expressing the modified polypeptide.

Attachment of Target Molecules to a Support

The methods can provide for conjugation of a polypeptide to a moiety tofacilitate attachment of the polypeptide to a solid substrate (e.g., tofacilitate assays), or to a moiety to facilitate easy separation (e.g.,a hapten recognized by an antibody bound to a magnetic bead). In someembodiments, the methods are used to provide for attachment of a proteinto an array (e.g., chip) in a defined orientation. For example, apolypeptide modified at a selected site (e.g., at or near theN-terminus) can be generated, and the methods, conjugates and compoundsused to deliver a moiety to the modified polypeptide. The moiety canthen be used as the attachment site for affixing the polypeptide to asupport (e.g., solid or semi-solid support, such as a support suitablefor use as a microchip in high-throughput assays).

Water-Soluble Polymers

In some cases, a conjugate includes a covalently linked water-solublepolymer. A moiety of particular interest is a water-soluble polymer. A“water-soluble polymer” refers to a polymer that is soluble in water andis usually substantially non-immunogenic, and usually has an atomicmolecular weight greater than 1,000 Daltons. The methods, conjugates andcompounds described herein can be used to attach one or morewater-soluble polymers to a polypeptide. Attachment of a water-solublepolymer (e.g., PEG) to a polypeptide, such as a pharmaceutically active(e.g., therapeutic) polypeptide can be desirable as such modificationcan increase the therapeutic index by increasing serum half-life as aresult of increased proteolytic stability and/or decreased renalclearance. Additionally, attachment of one or more polymers (e.g.,PEGylation) can reduce immunogenicity of protein pharmaceuticals.

In some embodiments, the water-soluble polymer has an effectivehydrodynamic molecular weight of greater than 5,000 Da, greater than10,000 Da, greater than 20,000 to 500,000 Da, greater than 40,000 Da to300,000 Da, greater than 50,000 Da to 70,000 Da, such as greater than60,000 Da. In some embodiments, the water-soluble polymer has aneffective hydrodynamic molecular weight of from 10 kDa to 20 kDa, from20 kDa to 25 kDa, from 25 kDa to 30 kDa, from 30 kDa to 50 kDa, or from50 kDa to 100 kDa. By “effective hydrodynamic molecular weight” isintended the effective water-solvated size of a polymer chain asdetermined by aqueous-based size exclusion chromatography (SEC). Whenthe water-soluble polymer contains polymer chains having polyalkyleneoxide repeat units, such as ethylene oxide repeat units, each chain canhave an atomic molecular weight of 200 Da to 80,000 Da, or 1,500 Da to42,000 Da, including 2,000 to 20,000 Da. Unless referred tospecifically, molecular weight is intended to refer to atomic molecularweight. Linear, branched, and terminally charged water soluble polymers(e.g., PEG) may be used.

Polymers useful as moieties to be attached to a polypeptide can have awide range of molecular weights, and polymer subunits. These subunitsmay include a biological polymer, a synthetic polymer, or a combinationthereof. Examples of such water-soluble polymers include: dextran anddextran derivatives, including dextran sulfate, P-amino cross linkeddextrin, and carboxymethyl dextrin, cellulose and cellulose derivatives,including methylcellulose and carboxymethyl cellulose, starch anddextrines, and derivatives and hydroylactes of starch, polyalkyleneglycol and derivatives thereof, including polyethylene glycol,methoxypolyethylene glycol, polyethylene glycol homopolymers,polypropylene glycol homopolymers, copolymers of ethylene glycol withpropylene glycol, wherein said homopolymers and copolymers areunsubstituted or substituted at one end with an alkyl group, heparin andfragments of heparin, polyvinyl alcohol and polyvinyl ethyl ethers,polyvinylpyrrolidone, aspartamide, and polyoxyethylated polyols, withthe dextran and dextran derivatives, dextrine and dextrine derivatives.It will be appreciated that various derivatives of the specificallyrecited water-soluble polymers are also contemplated.

Water-soluble polymers such as those described above includepolyalkylene oxide based polymers, such as polyethylene glycol “PEG”(See. e.g., “Poly(ethylene glycol) Chemistry: Biotechnical andBiomedical Applications”, J. M. Harris, Ed., Plenum Press, New York,N.Y. (1992); and “Poly(ethylene glycol) Chemistry and BiologicalApplications”, J. M. Harris and S. Zalipsky, Eds., ACS (1997); andInternational Patent Applications: WO 90/13540, WO 92/00748, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28937, WO 95/11924, WO 96/00080, WO 96/23794, WO 98/07713, WO98/41562, WO 98/48837, WO 99/30727, WO 99/32134, WO 99/33483, WO99/53951, WO 01/26692, WO 95/13312, WO 96/21469, WO 97/03106, WO99/45964, and U.S. Pat. Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992;5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838;5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001;5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805;5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662;5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990;5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900;5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455;5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042;6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095;6,194,580; 6,214,966).

Examples of polymers of interest include those containing a polyalkyleneoxide, polyamide alkylene oxide, or derivatives thereof, includingpolyalkylene oxide and polyamide alkylene oxide comprising an ethyleneoxide repeat unit of the formula —(CH₂—CH₂—O)—. Further examples ofpolymers of interest include a polyamide having a molecular weightgreater than 1,000 Daltons of the formula —[C(O)—X—C(O)—NH—Y—NH]n- or—[NH—Y—NH—C(O)—X—C(O)]_(n)—, where X and Y are divalent radicals thatmay be the same or different and may be branched or linear, and n is adiscrete integer from 2-100, such as from 2 to 50, and where either orboth of X and Y comprises a biocompatible, substantially non-antigenicwater-soluble repeat unit that may be linear or branched. Furtherexamples of water-soluble repeat units comprise an ethylene oxide of theformula —(CH₂—CH₂—O)— or —(CH₂—CH₂—O)—. The number of such water-solublerepeat units can vary significantly, with the number of such units beingfrom 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, for example from2 to 50. An example of an embodiment is one in which one or both of Xand Y is selected from: —((CH₂)_(n1)—(CH₂—CH₂—O)_(n2)—(CH₂)— or—((CH₂)_(n1)—(O—CH₂—CH₂)_(n2)—(CH₂)_(n-1)—), where n1 is 1 to 6, 1 to 5,1 to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2to 8, or 2 to 5. A further example of an embodiment is one in which X is—(CH₂—CH₂)—, and where Y is —(CH₂—(CH₂—CH₂—O)₃—CH₂—CH₂—CH₂)— or—(CH₂—CH₂—CH₂—(O—CH₂—CH₂)₃—CH₂)—.

The polymer can include one or more spacers or linkers. Examples ofspacers or linkers include linear or branched moieties comprising one ormore repeat units employed in a water-soluble polymer, diamino and ordiacid units, natural or unnatural amino acids or derivatives thereof,as well as aliphatic moieties, including alkyl, aryl, heteroalkyl,heteroaryl, alkoxy, and the like, which can contain, for example, up to18 carbon atoms or even an additional polymer chain.

The polymer moiety, or one or more of the spacers or linkers of thepolymer moiety when present, may include polymer chains or units thatare biostable or biodegradable. For example, polymers with repeatlinkages have varying degrees of stability under physiologicalconditions depending on bond lability. Polymers with such bonds can becategorized by their relative rates of hydrolysis under physiologicalconditions based on known hydrolysis rates of low molecular weightanalogs, e.g., from less stable to more stable, e.g., polyurethanes(—NH—C(O)—O—)>polyorthoesters (—O—C((OR)(R′))—O—)>polyamides(—C(O)—NH—). Similarly, the linkage systems attaching a water-solublepolymer to a target molecule may be biostable or biodegradable, e.g.,from less stable to more stable: carbonate (—O—C(O)—O—)>ester(—C(O)—O—)>urethane (—NH—C(O)—O—)> orthoester (—O—C((OR)(R′))—O—)>amide(—C(O)—NH—). In general, it may be desirable to avoid use of a sulfatedpolysaccharide, depending on the lability of the sulfate group. Inaddition, it may be less desirable to use polycarbonates and polyesters.These bonds are provided by way of example, and are not intended tolimit the types of bonds employable in the polymer chains or linkagesystems of the water-soluble polymers useful in the modified aldehydetagged polypeptides disclosed herein.

Formulations

The conjugates (including antibody conjugates) of the present disclosurecan be formulated in a variety of different ways. In general, where theconjugate is a polypeptide-drug conjugate, the conjugate is formulatedin a manner compatible with the drug conjugated to the polypeptide, thecondition to be treated, and the route of administration to be used.

The conjugate (e.g., polypeptide-drug conjugate) can be provided in anysuitable form, e.g., in the form of a pharmaceutically acceptable salt,and can be formulated for any suitable route of administration, e.g.,oral, topical or parenteral administration. Where the conjugate isprovided as a liquid injectable (such as in those embodiments where theyare administered intravenously or directly into a tissue), the conjugatecan be provided as a ready-to-use dosage form, or as a reconstitutablestorage-stable powder or liquid composed of pharmaceutically acceptablecarriers and excipients.

Methods for formulating conjugates can be adapted from those availablein the art. For example, conjugates can be provided in a pharmaceuticalcomposition comprising a therapeutically effective amount of a conjugateand a pharmaceutically acceptable carrier (e.g., saline). Thepharmaceutical composition may optionally include other additives (e.g.,buffers, stabilizers, preservatives, and the like). In some embodiments,the formulations are suitable for administration to a mammal, such asthose that are suitable for administration to a human.

Methods of Treatment

The polypeptide-drug conjugates of the present disclosure find use intreatment of a condition or disease in a subject that is amenable totreatment by administration of the parent drug (i.e., the drug prior toconjugation to the polypeptide). By “treatment” is meant that at leastan amelioration of the symptoms associated with the condition afflictingthe host is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g.symptom, associated with the condition being treated. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the condition, or at least the symptoms thatcharacterize the condition. Thus treatment includes: (i) prevention,that is, reducing the risk of development of clinical symptoms,including causing the clinical symptoms not to develop, e.g., preventingdisease progression to a harmful state; (ii) inhibition, that is,arresting the development or further development of clinical symptoms,e.g., mitigating or completely inhibiting an active disease; and/or(iii) relief, that is, causing the regression of clinical symptoms.

In the context of cancer, the term “treating” includes any or all of:reducing growth of a solid tumor, inhibiting replication of cancercells, reducing overall tumor burden, and ameliorating one or moresymptoms associated with a cancer.

The subject to be treated can be one that is in need of therapy, wherethe host to be treated is one amenable to treatment using the parentdrug. Accordingly, a variety of subjects may be amenable to treatmentusing the polypeptide-drug conjugates disclosed herein. Generally, suchsubjects are “mammals”, with humans being of interest. Other subjectscan include domestic pets (e.g., dogs and cats), livestock (e.g., cows,pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs,and rats, e.g., as in animal models of disease), as well as non-humanprimates (e.g., chimpanzees, and monkeys).

The amount of polypeptide-drug conjugate administered can be initiallydetermined based on guidance of a dose and/or dosage regimen of theparent drug. In general, the polypeptide-drug conjugates can provide fortargeted delivery and/or enhanced serum half-life of the bound drug,thus providing for at least one of reduced dose or reducedadministrations in a dosage regimen. Thus, the polypeptide-drugconjugates can provide for reduced dose and/or reduced administration ina dosage regimen relative to the parent drug prior to being conjugatedin an polypeptide-drug conjugate of the present disclosure.

Furthermore, as noted above, because the polypeptide-drug conjugates canprovide for controlled stoichiometry of drug delivery, dosages ofpolypeptide-drug conjugates can be calculated based on the number ofdrug molecules provided on a per polypeptide-drug conjugate basis.

In some embodiments, multiple doses of a polypeptide-drug conjugate areadministered. The frequency of administration of a polypeptide-drugconjugate can vary depending on any of a variety of factors, e.g.,severity of the symptoms, condition of the subject, etc. For example, insome embodiments, a polypeptide-drug conjugate is administered once permonth, twice per month, three times per month, every other week (qow),once per week (qw), twice per week (biw), three times per week (tiw),four times per week, five times per week, six times per week, everyother day (qod), daily (qd), twice a day (qid), or three times a day(tid).

Methods of Treating Cancer

The present disclosure provides methods for delivering a cancerchemotherapeutic agent to an individual having a cancer. The methods areuseful for treating a wide variety of cancers, including carcinomas,sarcomas, leukemias, and lymphomas.

Carcinomas that can be treated using a subject method include, but arenot limited to, esophageal carcinoma, hepatocellular carcinoma, basalcell carcinoma (a form of skin cancer), squamous cell carcinoma (varioustissues), bladder carcinoma, including transitional cell carcinoma (amalignant neoplasm of the bladder), bronchogenic carcinoma, coloncarcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma,including small cell carcinoma and non-small cell carcinoma of the lung,adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma,breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renalcell carcinoma, ductal carcinoma in situ or bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcarcinoma, uterine carcinoma, testicular carcinoma, osteogeniccarcinoma, epithelial carcinoma, and nasopharyngeal carcinoma, etc.

Sarcomas that can be treated using a subject method include, but are notlimited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma,rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be treated using a subject method include,but are not limited to, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Leukemias that can be treated using a subject method include, but arenot limited to, a) chronic myeloproliferative syndromes (neoplasticdisorders of multipotential hematopoietic stem cells); b) acutemyelogenous leukemias (neoplastic transformation of a multipotentialhematopoietic stem cell or a hematopoietic cell of restricted lineagepotential; c) chronic lymphocytic leukemias (CLL; clonal proliferationof immunologically immature and functionally incompetent smalllymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia,and hairy cell leukemia; and d) acute lymphoblastic leukemias(characterized by accumulation of lymphoblasts). Lymphomas that can betreated using a subject method include, but are not limited to, B-celllymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin'sB cell lymphoma; and the like.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any purificationprotocol known in the art, including chromatography, such as HPLC,preparative thin layer chromatography, flash column chromatography andion exchange chromatography. Any suitable stationary phase can be used,including normal and reversed phases as well as ionic resins. In certainembodiments, the disclosed compounds are purified via silica gel and/oralumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4^(th) edition, Vol. 15/1, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

The subject compounds can be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. A varietyof examples of synthetic routes that can be used to synthesize thecompounds disclosed herein are described in the schemes below.

Synthesis of HIPS-AF594

A hydrazinyl-indole detectable label conjugate (HIPS-AF594) wassynthesized as illustrated in Scheme 1, below:

Compound 20 (2-(trimethylsilyl)ethyl 1,2-dimethylhydrazinecarboxylate)was synthesized from Compound 10 as follows. To a vigorously stirredsuspension of sym-dimethylhydrazine dihydrochloride (7.5 mmol) in 15 mLMeCN was added triethylamine (18.8 mmol). The resulting white suspensionwas filtered and cooled to 0° C. While stirring,N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide (3.7 mmol) in 2 mLMeCN was added dropwise over 10 min. The reaction was allowed to warm toRT over 16 hr with stirring. The crude reaction was concentrated invacuo, then partitioned between 100 mL EtOAc and 30 mL water. Theorganic phase was washed 3×30 mL brine, dried over MgSO₄, filtered andconcentrated in vacuo to afford the product in a 64% yield. The productrequired no further purification. ¹H NMR (400 MHz, CDCl₃) δ 4.5 (bs,1H), 4.20 (dd, J=8.8, 8.4 Hz, 2H), 3.03 (s, 3H), 2.57 (s, 3H), 1.02 (dd,J=8.8, 8.4 Hz, 2H), 0.04 (s, 9H).

Compound 40 (2-(trimethylsilyl)ethyl2-((1-(3-methoxy-3-oxopropyl)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazinecarboxylate)was synthesized from Compound 20 and Compound 30 as follows. An ovendried 4 mL vial was charged with Compound 30 (methyl3-(2-formyl-1H-indol-1-yl)propanoate; 0.0606 mmol), Compound 20(2-(trimethylsilyl)ethyl 1,2-dimethylhydrazinecarboxylate; 0.0828 mmol),acetic acid (0.0727 mmol) in 150 μL anhydrous methanol. Sodiumcyanoborohydride (0.0727 mmol) was added in 100 μL anhydrous methanoland stirred for 16 hr. The crude reaction mixture was purified bypreparatory TLC (500 m thickness, silica gel) using a mobile phase of15% EtOAc:Hexanes to afford the product in a 45% yield. ¹H NMR (400 MHz,CDCl₃) δ7.57 (d, J=7.6 Hz, 1H), 7.35 (d, J=8.4 Hz, 1H), 7.22 (ddd,J=8.0, 7.2, 1.2 Hz, 1H), 7.10 (ddd, J=8.0, 7.2, 1.2 Hz, 1H), 6.40 (s,1H), 4.65 (bs, 2H), 4.26-4.11 (m, 4H), 3.70 (s, 3H), 2.90 (m, 5H), 2.66(s, 3H), 0.99 (t, J=8.4, 2H), 0.05 (s, 9H).

Compound 50(3-(2-((1,2-dimethyl-2-((2-(trimethylsilyl)ethoxy)carbonyl)hydrazinyl)methyl)-1H-indol-1-yl)propanoicacid; also referred to as Teoc-HIPS—OH) was synthesized from Compound 40as follows. A 4 mL vial was charged with Compound 40(2-(trimethylsilyl)ethyl2-((1-(3-methoxy-3-oxopropyl)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazinecarboxylate;0.027 mmol), lithium hydroxide (0.054 mmol) and 250 μL of methanol andstirred for 24 h. The solution was concentrated in vacuo, thenchromatographed in 1% AcOH:EtOAc to yield the desired product in an 89%yield. ¹H NMR (400 MHz, CDCl₃) δ7.57 (d, J=7.6 Hz, 1H), 7.38 (d, J=8.0Hz, 1H), 7.24 (ddd, J=8.0, 7.6, 0.8 Hz, 1H), 7.12 (ddd, J=7.6, 7.6, 0.8Hz, 1H), 6.40 (s, 1H), 4.63 (bs, 2H), 4.20 (t, J=8.4, 2H), 4.13 (s, 2H),3.11-2.97 (m, 5H), 2.58 (s, 3H), 2.66 (s, 3H), 0.99 (t, J=8.4, 2H), 0.05(s, 9H). LRMS (ESI) calcd for C₂₀H₃₀N₃O₄Si [M+H]⁺: 406.22; found 405.9.

Compound 70 (Teoc-HIPS-AF594) was synthesized from Compound 50(Teoc-HIPS—OH) and Compound 60 (AF594) as follows. To a solution ofCompound 60 (AF594; 0.00124 mmol), Compound 50 (Teoc-HIPS-OH; 0.0059mmol), triethylamine (0.0059 mmol) in 200 μL 1:1 DCM:DMF was added HATU((dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminiumhexafluorophosphate; 0.0031). The reaction was stirred for 4 h. Thecrude reaction was mixture was purified by flash chromatography (C₁₈)using a 0-33% MeCN:water gradient. LRMS (ESI) calcd for C₆₁H₇₇N₇O₁₂S₂Si[M+H]⁺: 1192.49; found 1194.1.

Compound 80 (HIPS-AF594) was synthesized from Compound 70(Teoc-HIPS-AF594) as follows. To a solution of Compound 70(Teoc-HIPS-AF594; 0.67 μmol) in 100 μL DMF was added 1 mg of CsF (6.6μmol) and heated to 60° C. for 2 hr. The reaction was then cooled toroom temperature and chromatographed (C18) with a gradient of 0-50%MeCN:water to yield 0.4 mg of a mixture of desired product and desiredproduct+CH₂ (addition of formaldehyde). LRMS (ESI) calcd forC₅₄H₆₃N₇O₁₁S₂[M+H]⁺: 1050.41; found 1151.4.

Synthesis of Fmoc-HIPS-OPFP

Fmoc-HIPS-OPFP ((9H-fluoren-9-yl)methyl1,2-dimethyl-2-((1-(3-oxo-3-(perfluorophenoxy)propyl)-1H-indol-2-yl)methyl)hydrazinecarboxylate)was synthesized as illustrated in Scheme 2, below:

Compound 100 (methyl 3-(2-formyl-1H-indol-1-yl)propanoate) wassynthesized from Compound 90 as follows. To a solution of Dess-MartinPeriodinane (1.2 mmol) in 5 mL DCM was added 3 mmol of pyrindine andsonicated until soluble, whereupon 1 mmol of Compound 90 (methyl3-(2-(hydroxymethyl)-1H-indol-1-yl)propanoate) was added and thesolution stirred for 2.5 h. The reaction mixture was then concentratedand chromatographed in DCM to afford 0.76 mmol of product. ¹H NMR (400MHz, CDCl₃) δ 9.90 (s, 1H), 7.75 (d, J=8.4 Hz, 1H), 7.53 (dd, J=8.4, 0.8Hz, 1H), 7.45 (ddd, J=8.0, 6.8, 0.8 Hz, 1H), 7.32 (d, J=0.8 Hz, 1H),7.19 (ddd, J=8.0, 6.8, 0.8 Hz, 1H), 4.86 (t, J=7.2 Hz, 2H), 3.64 (s,3H), 2.86 (t, J=7.2 Hz, 2H).

Compound 110 (3-(2-formyl-1H-indol-1-yl)propanoic acid) was synthesizedfrom Compound 100 as follows. To a solution of 1.03 mmol of Compound 100(methyl 3-(2-formyl-1H-indol-1-yl)propanoate) in 5 mL methanol was added3.09 mmol of lithium hydroxide and stirred for 5 h, upon which thesolution was acidified to pH 2 and partitioned between 5 mL water and 15mL ethyl acetate. The aqueous phase was washed 2×10 mL ethyl acetate.The combined organic phases were washed 1×10 mL Brine, then dried overmagnesium sulfate, filtered and concentrated by rotary evaporation toyield 1.01 mmol of product. The crude material was pure by NMR and takenon to the subsequent reaction. ¹H NMR (400 MHz, CDCl₃) δ 9.89 (s, 1H),7.76 (d, J=8.4 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H), 7.45 (ddd, J=8.4, 6.8,1.2 Hz, 1H), 7.33 (s), 7.22 (dd, J=6.8, 0.8 Hz, 1H), 4.86 (t, J=7.2 Hz,2H), 2.91 (t, J=7.2 Hz, 2H).

Compound 120 ((9H-fluoren-9-yl)methyl 1,2-dimethylhydrazinecarboxylate)was synthesized from Compound 10 according to literature procedure asdescribed in Nicolas, I. et al. Synlett. 2011, 3, 327-330, thedisclosure of which is incorporated herein by reference.

Compound 130(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoicacid) was synthesized from Compound 110 and Compound 120 as follows. A50 mL flame dried round bottom flask was charged with Compound 110(3-(2-formyl-1H-indol-1-yl)propanoic acid; 3.22 mmol), Compound 120((9H-fluoren-9-yl)methyl 1,2-dimethylhydrazinecarboxylate; 5.0 mmol) anddissolved in 26 mL dichloroethane (DCE). To this flask, sodiumtriacetoxyborohydride (3.54 mmol) was added portionwise and stirred forthree hours then quenched with 5 mL 1M NaOH (aq) and stirred for 5 min.The reaction was acidified to pH 2 and partitioned with 50 mL DCM. Theorganic phase was extracted three times with 10 mL 5% citric acid (aq),then dried over MgSO₄, filtered and concentrated in vacuo to afford 55%of the desired product.

Compound 140 ((9H-fluoren-9-yl)methyl1,2-dimethyl-2-((1-(3-oxo-3-(perfluorophenoxy)propyl)-1H-indol-2-yl)methyl)hydrazinecarboxylate;also referred to as Fmoc-HIPS-OPFP) was synthesized from Compound 130 asfollows. A flame dried round bottom flask was charged with Compound 130(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoicacid; 0.574 mmol), pentafluorophenol (PFP; 0.631 mmol), 5 mL ethylacetate (dry) and then cooled to 0° C. To this solution was addedN,N′-dicyclohexylcarbodiimide (0.631 mmol) in 0.5 mL EtOAc and allowedto warm to 20° C. with stirring. Upon consumption of starting materialby TLC (˜2 h), the crude reaction was diluted in 60 mL EtOAc, washed2×20 mL water, 2×20 mL brine, dried over MgSO₄, filtered andconcentrated in vacuo. This material was used in subsequent reactionswithout further purification.

Synthesis of HIPS-6PEG-Maytansine

HIPS-6PEG-Maytansine was synthesized as illustrated in Scheme 3, below:

Compound 150 (Fmoc-HIPS-6PEG-OH) was synthesized from Compound 140(Fmoc-HIPS-OPFP) as follows. A 1.6 mL vial was charged with Compound 140(Fmoc-HIPS-OPFP; 66.6 mg, 0.1 mmol),1-amino-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (74.9 mg, 0.2 mmol),DIPEA (79.5 mg, 107.1 μL, 0.6 mmol) and 1 mL of anhydrous DMF. Thereaction was stirred for 2 h and then chromatographed (C18) with agradient of 0-100% MeCN:water to afford the product as a white film(48.6 mg, 58% yield).

Compound 170 was synthesized from Compound 160, as shown in the schemebelow, according to literature procedure as described inPCT/US2006/030857 (published as WO 2007/021674), the disclosure of whichis incorporated herein by reference.

Compound 180 (Fmoc-HIPS-6PEG-deacyl-maytansine) was synthesized fromCompound 150 (Fmoc-HIPS-6PEG-OH) and Compound 170 as follows. A 1.6 mLvial was charged with Compound 150 (Fmoc-HIPS-6PEG-OH; 18.5 mg, 0.023mmol), HATU (8.8 mg, 0.023 mmol), DIPEA (8.8 mg, 11.9 uL, 0.068 mmol),0.1 mL anhydrous DMF and allowed to stir for 15 min at room temperature.To this was added Compound 170 (N-Methylalanine maytansine; 14.5 mg,0.022 mmol) in 0.1 mL anhydrous DMF. The reaction was stirred for 2 h,added to 2 mL water, and extracted with 5×1 mL EtOAc. The organic phasewas washed with 1×1 mL H₂O, 1×1 mL 1 M HCl, 1×1 mL 1.2 M NaHCO₃, 1×1 mLH₂O, 1×1 mL 5M NaCl, and dried over Na₂SO₄. The product waschromatographed (silica) using a gradient of 0-10% MeOH:CH₂Cl₂ to affordthe desired compound as a pale yellow film (19.9 mg, 62% yield).

Compound 190 (HIPS-6PEG-Maytansine) was synthesized from Compound 180(Fmoc-HIPS-6PEG-deacyl-maytansine) as follows. A 1.6 mL vial was chargedwith Compound 180 (Fmoc-HIPS-6PEG-deacyl-maytansine; 19.9 mg, 0.014mmol), piperidine (116.8 mg, 135.5 uL, 1.37 mmol), and 0.68 mL DMA. Uponconsumption of the starting material, as indicated by HPLC,approximately 20 min, the crude reaction was immediately chromatographed(C18) using a 0-100% MeCN:water gradient to afford the desired productas a white film (10.2 mg, 61% yield).

Example 1

Conjugation of HIPS-A594 to an Aldehyde-Tagged IgG Antibody

HIPS-AF594 (Compound 80, above) was conjugated to an aldehyde-taggedantibody over a range of time and pH conditions. HIPS-AF594 (92.6drug:aldehyde equivalents) was reacted with 5.4 μM aldehyde-tagged IgGantibody for 1, 2, 3, 4, or 24 hr at 37° C. in PBS pH 7.4, PBS with 100mM potassium acetate pH 4.6, PBS with 50 mM sodium citrate pH 6.0, orPBS with 100 mM Tris pH 8.

Results of the conjugation reaction are shown in FIG. 2 , panels A-C,which shows conjugation of HIPS-AF594 to aldehyde-tagged antibodyconjugated over a range of pH conditions. FIG. 2 , panel A, shows areaction scheme for the synthesis of the functionalized detectablelabel. FIG. 2 , panel B, shows a schematic of the conjugation reactionof the functionalized detectable label to the antibody. FIG. 2 , panelC, shows images of SDS-PAGE gels, which were used to analyze theconjugate. The presence of conjugate was determined by fluorescenceimaging.

Example 2

Stability of Antibody-Detectable Label Conjugate

HIPS-AF594 (Compound 80, above) (42.4 drug:aldehyde equivalents) wasreacted with 5.9 μM aldehyde-tagged IgG antibody for 16 hr at 37° C. in128 mM sodium chloride and 50 mM sodium citrate pH 6. The unreactedHIPS-AF594 was removed from the reaction mixture by size exclusionchromatography. The resulting antibody-fluorophore conjugate wasincubated at 4° C. or 37° C. in either plasma or serum (both human andmouse) for 1, 3 or 6 days. SDS-PAGE was used to analyze the conjugate.The presence of conjugate was determined by fluorescence imaging. Asshown in FIG. 3 , no free dye was observed in either case and theconjugate had not decomposed.

Results of the conjugation reaction are shown in FIG. 3 , which showsimages of SDS-PAGE gels showing the stability of an antibody-detectablelabel conjugate over a 6 day time period.

Example 3

Conjugation of HIPS-6PEG-Maytansine to an Aldehyde-Tagged Antibody

HIPS-6PEG-Maytansine (Compound 190, above; 231.5 drug:aldehydeequivalents) was reacted with 5.4 uM aldehyde-tagged antibody (A) in PBSwith 50 mM sodium citrate pH 6 and 10% DMA at 37° C. The reactionmixture was analyzed after 48 hours.

Results are shown in FIG. 4 , which shows a hydrophobic interactioncolumn (HIC) trace of the aldehyde-tagged antibody conjugated toHIPS-6PEG-Maytansine. The unconjugated (A), mono-conjugate (B), anddi-conjugate (C) protein conjugate were observed.

Additional compounds were synthesized as described below.

General Synthetic Methods

All reagents were obtained from Sigma-Aldrich, Acros, or TCI and usedwithout further purification except piperidine, which was dried overCaH₂ and distilled. Anhydrous solvents were obtained from commercialsources in sealed bottles. Column chromatography was performed with aBiotage Isolera Prime chromatograph. High-pressure liquid chromatographywas performed on an Agilent 1100 chromatograph equipped with an AgilentPoroshell 120 column (4.6×150 mm), with UV absorption monitored at 205nm.

NMR spectra were acquired on a Bruker 400 MHz spectrometer by EmeryvillePharmaceutical Services. ¹H NMR spectra were referenced to residualCHCl₃ (7.26 ppm), CD₂HCN (1.94 ppm), or CD₂HOD (3.31 ppm). ¹³C NMRspectra were referenced to CDCl₃ (77.16 ppm), CD₃CN (1.32 ppm), or CD₃OD(49.00 ppm). NMR spectra were processed using MestReNova (MestrelabResearch S.L.). High-resolution ESI mass spectra of small molecules wereobtained at the UC Berkeley Mass Spectrometery Facility on a Thermo LTQOrbitrap mass spectrometer.

HIPS ligation reagent (Compound 4) was synthesized as illustrated inScheme 4, below:

Synthesis of Methyl 3-(2-formyl-1H-indol-1-yl)propanoate (1)

Compound 1 was synthesized as follows. Dess-Martin periodinane (5.195 g,12.25 mmol, 1.09 equiv) was suspended in a mixture of dichloromethane(20 mL) and pyridine (2.70 mL, 33.5 mmol, 3.0 equiv). After 5 min, theresulting white suspension was transferred to a solution of methyl3-(2-(hydroxymethyl)-1H-indol-1-yl)propanoate (2.611 g, 11.19 mmol) indichloromethane (10 mL), resulting in a red-brown suspension. After 1 h,the reaction was quenched with sodium thiosulfate (10% aqueous solution,5 mL) and sodium bicarbonate (saturated aqueous solution, 5 mL). Theaqueous layer was extracted with dichloromethane (3×20 mL), then driedover sodium sulfate, filtered, and concentrated to a brown oil.Purification by silica gel chromatography (5-50% ethyl acetate inhexanes) yielded the product as a colorless oil (2.165 g, 9.363 mmol,84%). ¹H NMR (400 MHz, CDCl₃) δ 9.87 (s, 1H), 7.73 (dt, J=8.1, 1.0 Hz,1H), 7.51 (dd, J=8.6, 0.9 Hz, 1H), 7.45-7.40 (m, 1H), 7.29 (d, J=0.9 Hz,1H), 7.18 (ddd, J=8.0, 6.9, 1.0 Hz, 1H), 4.84 (t, J=7.2 Hz, 2H), 3.62(s, 3H), 2.83 (t, J=7.2 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 182.52,171.75, 140.12, 135.10, 127.20, 126.39, 123.46, 121.18, 118.55, 110.62,51.83, 40.56, 34.97. HRMS (ESI) calcd for C₁₃H₁₃NO₃Na [M+Na]⁺: 254.0793;found: 254.0786.

Synthesis of 3-(2-formyl-1H-indol-1-yl)propanoic acid (2)

Compound 2 was synthesized from Compound 1 as follows. To a solution ofCompound 1 (306 mg, 1.32 mmol) dissolved in dioxane (18 mL) was addedLiOH (2 M aqueous solution, 992 μL, 1.98 mmol, 1.5 equiv). After 1 h,hydrochloric acid (1 M aqueous solution) was added dropwise to give asolution with pH=5. The solution was concentrated and the resulting palebrown oil was dissolved in ethyl acetate (20 mL) and washed with aceticacid (5% aqueous solution, 10 mL), water (10 mL), and brine (10 mL). Theorganic layer was dried over sodium sulfate, filtered, and concentratedto a pale brown oil. Purification by silica gel chromatography (5-50%ethyl acetate in hexanes with 0.1% acetic acid) yielded the product as apale yellow solid (290 mg, 1.34 mmol, quantitative). ¹H NMR (400 MHz,CDCl₃) δ 9.89 (s, 1H), 7.76 (dt, J=8.1, 0.9 Hz, 1H), 7.53 (dd, J=8.6,0.9 Hz, 1H), 7.48-7.43 (m, 1H), 7.33 (d, J=0.8 Hz, 1H), 7.21 (ddd,J=8.0, 6.9, 1.0 Hz, 1H), 4.85 (t, J=7.2 Hz, 2H), 2.91 (t, J=7.2 Hz, 2H).¹³C NMR (101 MHz, CDCl₃) δ 182.65, 176.96, 140.12, 135.02, 127.33,126.42, 123.53, 121.27, 118.76, 110.55, 40.19, 34.82. HRMS (ESI) calcdfor C₁₂H₁₀NO₃ [M−H]⁻: 216.0666; found: 216.0665.

Synthesis of3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoicacid (3)

Compound 3 was synthesized from Compound 2 as follows. To a solution ofCompound 2 (1.193 g, 5.492 mmol) and FmocN(Me)NHMe (2.147 g, 7.604 mmol,1.38 equiv) in 1,2-dichloroethane (anhydrous, 25 mL) was added sodiumtriacetoxyborohydride (1.273 g, 6.006 mmol, 1.09 equiv). The resultingyellow suspension was stirred for 2 h and then quenched with sodiumbicarbonate (saturated aqueous solution, 10 mL), followed by addition ofhydrogen chloride (1 M aqueous solution) to pH 4. The organic layer wasseparated, and the aqueous layer was extracted with dichloromethane(5×10 mL). The pooled organic extracts were dried over sodium sulfate,filtered, and concentrated to an orange oil. Purification by C18 silicagel chromatography (20-90% acetonitrile in water) yielded 3 as a waxypink solid (1.656 g, 3.425 mmol, 62%). ¹H NMR (400 MHz, CDCl₃) δ 7.76(d, J=7.4 Hz, 2H), 7.70-7.47 (br m, 3H), 7.42-7.16 (br m, 6H), 7.12-7.05(m, 1H), 6.37 (s, 0.6H), 6.05 (s, 0.4H), 4.75-4.30 (br m, 4H), 4.23 (m,1H), 4.10 (br s, 1H), 3.55 (br d, 1H), 3.11-2.69 (m, 5H), 2.57 (br s,2H), 2.09 (br s, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 174.90, 155.65, 143.81,141.42, 136.98, 134.64, 127.75, 127.48, 127.12, 124.92, 122.00, 120.73,120.01, 119.75, 109.19, 103.74, 67.33, 66.80, 51.39, 47.30, 39.58,39.32, 35.23, 32.10. HRMS (ESI) calcd for C₂₉H₃₀N₃O₄ [M+H]⁺: 484.2236;found: 484.2222.

Synthesis of Piperidinium3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoate (4)

Compound 4 was synthesized from Compound 3 as follows. Indole 3 (64.2mg, 133 μmol) was dissolved in a solution of piperidine in inN,N-dimethylacetamide (20% v/v, 1.31 mL, 2.65 mmol, 20.0 equiv). After20 min, the solution was purified by C18 silica gel chromatography(0-100% acetonitrile in water). The product was isolated as a colorlessoil (28.4 mg, 82.0 μmol, 62%). ¹H NMR (400 MHz, 7:5 D₂O:CD₃CN) δ 7.84(d, J=7.9 Hz, 1H), 7.76 (d, J=8.3 Hz, 1H), 7.49 (m, 1H), 7.36 (m, 1H),6.73 (s, 1H), 4.73-4.69 (m, 2H), 4.25 (s, 2H), 3.36-3.29 (m, 4H),2.86-2.80 (m, 2H), 2.78 (s, 3H), 2.67 (s, 3H), 1.99-1.95 (m, 4H),1.88-1.83 (m, 2H). ¹³C NMR (151 MHz, 7:5 D₂O:CD₃CN) δ 179.80, 137.61,135.78, 128.40, 122.59, 121.29, 120.50, 110.94, 103.70, 53.86, 45.32,42.79, 41.43, 38.64, 33.95, 23.07, 22.41, 1.32. HRMS (ESI) calcd forC₁₄H₂₀N₃O₂ [M-piperidine+H]⁺: 262.1556; found: 262.1547.

Synthesis of3-(1-((benzyloxy)methyl)-2,3-dimethyl-3,4-dihydro-1H-pyridazino[4,5-b]indol-5(2H)-yl)propanoicacid (5)

Compound 4 was reacted with benzyloxyacetaldehyde, which was chosen as amodel small molecule aldehyde for its UV absorption and aqueoussolubility properties, to produce the azacarboline product (Compound 5).To a solution of Compound 4 (16.2 mg, 46.8 μmol) in aqueous acetonitrile(1:3 water:acetonitrile, 800 μL) was added benzyloxyacetaldehyde (7.23μL, 51.5 mol, 1.10 equiv). After 1 h, the solution was purified by C18silica gel chromatography (0-100% acetonitrile in water with 0.1% aceticacid). Toluene was added to the eluent to aid in removal of residualacetic acid, and the product was isolated as a pale brown oil (16.2 mg,41.2 μmol, 88%). ¹H NMR (400 MHz, CDCl₃) δ 7.41 (d, J=7.8 Hz, 1H),7.38-7.34 (m, 2H), 7.34-7.30 (m, 3H), 7.18-7.15 (m, 2H), 7.08-7.05 (m,1H), 4.65 (d, J=12.4 Hz, 1H), 4.58 (d, J=12.4 Hz, 1H), 4.28-4.18 (m,2H), 4.10 (m, 1H), 3.91 (t, J=9.0 Hz, 1H), 3.81 (s, 1H), 3.76-3.64 (m,2H), 2.69 (t, J=6.8 Hz, 2H), 2.63 (s, 3H), 2.39 (s, 3H). ¹³C NMR (151MHz, CDCl₃) δ 175.08, 138.74, 136.27, 131.75, 128.42, 127.92, 127.60,126.61, 121.43, 119.61, 118.74, 109.33, 105.41, 73.46, 71.94, 61.61,44.16, 41.33, 39.19, 34.98, 31.17. HRMS (ESI) calcd for C₂₃H₂₈N₃O₃[M+H]⁺: 394.2131; found: 394.2120.

Synthesis of Fmoc-HIPS-PFP

Fmoc-HIPS-PFP was synthesized as follows. To a solution of Fmoc-HIPSacid (Compound 4) (1.4673 g, 3.0344 mmol) and pentafluorophenol (608.1mg, 3.304 mmol, 1.09 equiv) in ethyl acetate (anhydrous, 10 mL) at 0° C.was added a solution of N,N′-dicyclohexylcarbodiimide (684.0 mg, 3.315mmol, 1.09 equiv) in ethyl acetate (anhydrous, 15 mL). A whiteprecipitate began to form and after 5 min the solution was allowed towarm to room temperature. After 90 min, the suspension was cooled to 0°C. and filtered through Celite; the filter cake was washed with coldethyl acetate (5 mL). The filtrate was washed with water (4×10 mL) andbrine (10 mL), dried over sodium sulfate, filtered, and concentrated toa pale yellow solid (1.979 g, 3.046 mmol, 100%). This material was usedin subsequent reactions without further purification.

Synthesis of Fmoc-HIPS-AF488

Fmoc-HIPS-AF488 was synthesized as follows. To a solution ofFmoc-HIPS-PFP (3.0 mg) and Alexa Fluor 488 (AF488) cadaverine (1 mg, 1.6μmol) in N,N-dimethylfonnamide (0.2 mL) was added sodium carbonate (100mM aqueous solution, 31.2 μL, 3.12 □mol). After 2 h, the reactionmixture was concentrated to a red-orange oil which was purified by C18silica gel chromatography (H₂O/MeCN). The product was isolated as a redsolid (1.7 mg, 1.54 μmol, 99%). HRMS (ESI) calcd forC₅₅H₅₂N₇O₁₃S₂[M−Na]⁻: 1082.3070; found: 1082.3064.

Synthesis of HIPS-AF488 Ligation Reagent (Compound 9)

HIPS-AF488 ligation reagent (Compound 9) was synthesized as follows.Fmoc-HIPS-AF488 (1.7 mg, 1.54 μmol) was dissolved in a solution ofdioxane: MeOH: 2 M aqueous NaOH (30:9:1, 94 μL, 4.7 μmol NaOH, 3.1 equivNaOH). After 30 min, the pale blue reaction mixture was quenched withacetic acid (5% aqueous solution, 50 μL), resulting in a fluorescentgreen solution which was concentrated to a red solid. The product waspurified by C18 silica gel chromatography and further purified by HPLCon an Agilent Zorbax 300SB-C18 column (9.4×250 mm). The product wasisolated as a red solid (1.0 mg, 1.16 μmol, 75%). HRMS (ESI) calcd forC₄₀H₄₂N₇O₁₁S2 [M−Na]⁻: 860.2389; found: 860.2387.

Example 4

Reactivity of HIPS ligation reagent (Compound 4)

Experiments were performed to study the reactivity of HIPS reagent(Compound 4) relative to model aminooxy, hydrazide, and Pictet-Spenglerligation reagents with benzyloxyacetaldehyde (FIG. 5 , panels A and B,and FIG. 6 , panels A-D).

Experimental Protocol for HPLC Kinetics Assay: Reaction mixturescontained amine 4, 6, 7, or 8 (50 μM), benzyloxyacetaldehyde (50 μM),bromocresol green (10 μM), and buffer (10 mM) in a 120 μL aqueoussolution containing 6.7% MeCN, which was present to solubilize the stocksolution (1 mM) of benzyloxyacetaldehyde. After 2 h at room temperature,a 100 μL aliquot was analyzed by HPLC, employing a gradient of 10 to100% acetonitrile in water over 18 min.

As described above, buffered solutions containing 50 μM amine andaldehyde were incubated at room temperature for 2 h prior to analysis byHPLC. Within a pH range encompassing conditions commonly used foraldehyde and ketone bioconjugation reactions, aminooxy indole 6 (a modelPictet-Spengler ligation reagent) and model aminooxy compound 7 reactedmore quickly under acidic conditions. The reaction of hydrazide 8 didnot proceed appreciably under any conditions, which may be due to thelow equilibrium constant for hydrazone formation. Above pH 6, indole 4had a higher % conversion to product than the other amine nucleophiles,which indicated that the HIPS ligation may be useful for labelingproteins near neutral pH. A bell-shaped pH-rate curve was observed forthe reaction with Compound 4; the decreased reaction rate at lower pHmay be due to rate-limiting carbonyl addition due to protonation of thehydrazine moiety of Compound 4, since trialkylhydrazines such as the onein Compound 4 (pKa˜6.56) are more basic than O-alkylaminooxy moietiessuch as the one in Compound 6 (pKa˜4.60).

Example 5

Conjugation of Compound 4 to FGly-MBP

Experiments were performed to selectively label an aldehyde-containingprotein (FGly-MBP) with Compound 4 at near-neutral pH.

Experimental Protocol for Conjugation of 4 to FGly-MBP: Indole 4 (1 mM)and FGly-MBP (1.41 mg/mL) were combined in sodium citrate buffer (50 mM,pH 5.0) and incubated at 37° C. for 24 h. Excess Compound 4 was removedby buffer exchange into PBS in a centrifugal concentrator (Amicon, 10kDa MWCO) and the sample was then treated with trypsin (1.1 ug, 2 wt %)and incubated at 37° C. for 24 h. The peptides were desalted using a C18column (The Nest Group), eluting with 70% acetonitrile, 1% formic acidin water. The eluent was concentrated by centrifugal evaporation andthen analyzed by nano-LC/MS at the U.C. Berkeley QB3/Chemistry MassSpectrometry Facility on a Waters Q-Tof premier electrospray ionizationtime of flight mass spectrometer connected to a Waters nanoAcquity UPLC.

An aldehyde-tagged maltose binding protein (FGly-MBP), which is avariant of MBP that bears a Ca-formylglycine (FGly) residue near its Cterminus, was used as a substrate. As described previously, theformylglycine residue was installed by inclusion of the short peptidesequence LCTPSR at the C-terminus of the protein. Coexpression of MBPwith the Mycobacterium tuberculosis formylglycine generating enzymeresulted in cotranslational oxidation of the consensus sequence cysteineresidue to formylglycine, which was selectively reactive with thehydrazine-containing reagent 4. FGly-MBP was reacted with indole 4overnight and then the resulting conjugate was trypsinized. Analysis ofthe tryptic digest by ESI-MS showed that Compound 4 reacted with theFGly residue.

Example 6

Protein Labeling Experiments

Experiments were performed to determine the speed of the HIPS ligationunder mild reaction conditions by assessing labeling kinetics onproteins at pH 6.0.

Experimental Protocol for Protein Labeling Experiments: Reactionmixtures contained fluorophore 9, 10, 11, or 12 (400 μM) and protein(MBP, 0.67 mg/mL; α-HER2, 0.37 mg/mL; or Mb, 0.51 mg/mL) in sodiumphosphate buffer (100 mM, pH 6.0). After 2 h at 37° C., the reaction wasstopped by adding Tris-Cl to pH 8.5-9 and immediately analyzed bySDS-PAGE. Since the free fluorophores coeluted with and obscured Mb, theprotein was wet transferred to a nitrocellulose membrane to remove freefluorophore prior to fluorescence imaging, and protein loading wasassessed using Ponceau S.

To facilitate the analysis of protein conjugation experiments bySDS-PAGE, a fluorophore-functionalized HIPS indole (Compound 9) wasprepared by coupling Compound 3 with Alexa Fluor 488 cadaverine followedby Fmoc deprotection with piperidine. In these experiments, the relativeconjugation efficiencies of HIPS reagent 9 were compared to both thecommercially-available AF488 hydrazide (Compound 10) and the aminooxyreagent (Compound 11). This experimental design allowed for a comparisonof the HIPS ligation to the previously-reported Pictet-Spengler ligationas well as commercially-available aminooxy AF488 (Compound 12, FIG. 7 ,panel A). The relative labeling of two formylglycine-bearing proteinswas assessed at pH 6.0: FGly-MBP and FGly-α-HER2, a variant of thetherapeutic monoclonal antibody Herceptin that contains a formylglycineresidue at the C-terminus of each of its heavy chains. To show thegenerality of the method with an aldehyde other than formylglycine, thelabeling of chemically modified myoglobin (Mb) containing an N-terminalglyoxamide installed by pyridoxal phosphate-mediated transamination wasalso assessed. In all three cases, treatment of thealdehyde-functionalized protein with 400 μM of the fluorophore reagents9-12 at pH 6.0 for 2 h showed that labeling with HIPS reagent 9 wasfaster than labeling with the aminooxy and hydrazido-fluorophore panel(FIG. 7 , panels B-D). Control experiments with the FGly-MBP C390Amutant (which lacked the requisite cysteine residue for conversion toformylglycine), α-HER2 without the aldehyde tag sequence, and wild-typeMb, all of which lack aldehyde functionality, showed negligible labelingwith Compound 9. The experiment using FGly-α-HER2 demonstrated a methodfor the site-specific conjugation of a monoclonal antibody with a smallmolecule under mild conditions.

Example 7

Hydrolytic Stability Experiments

Experiments were performed to determine the hydrolytic stability of thelinkage on FGly-MBP relative to oximes, which are typicallyhydrolytically stable linkages for aldehydes and ketones inbioconjugation chemistry.

Experimental Protocol for MBP-AF488 Conjugate Hydrolysis ELISA

Preparation of MBP-AF488 Conjugates: Fluorophore 9 or 12 (545 μM) andFGly-MBP (1.61 mg/mL, 40 μg) were combined in sodium citrate buffer (45mM, pH 4.5). After 18 h at 37° C., the proteins were purified away fromfree fluorophore by ion-exchange chromatography (GE HiTrap SPXL, 5 mM to1 M NaCl in 5 mM sodium citrate pH 5.5). Conjugation of the AF488 dyewas verified by UV-vis spectroscopy.

Incubation of Conjugates in Human Plasma: A solution of MBP-AF488conjugate (10 μg/mL) in human plasma (0.98×) was incubated at 37° C. for5 d. Aliquots were withdrawn at 10-14 h intervals and diluted with 1%BSA in PBS to 10 ng/mL; the resulting solutions were immediately frozenat −80° C. until analysis by ELISA.

ELISA: A 96-well microtiter plate (Nunc Maxisorp) was coated with α-MBP(2.5 g/mL in PBS, Abcam) at 4° C. overnight. The wells were washed twicewith PBST, then incubated with BSA (1% in PBS) for 2 h. The wells werewashed twice with PBST, then incubated with MBP-AF488 conjugatesolutions (10 ng/mL). After 1 h, the wells were washed four times withPBST, then incubated with rabbit α-AF488 IgG (Invitrogen, 1:5000) for 1h. The wells were washed four times with PBST, then incubated withHRP-conjugated donkey α-rabbit IgG (Jackson ImmunoResearch, 1:10000) for1 h. After washing the wells four times with PBST, tetramethylbenzidineand hydrogen peroxide (Pierce TMB kit) were added and the reaction wasquenched by addition of 1 volume of H₂SO₄ (2 M) after 15 min. Absorptionwas read at 450 nm on a plate reader (SpectraMax M5).

As described above, purified azacarboline- and oxime-linked AF488conjugates of FGly-MBP were incubated at 10 μg/mL in human plasma at 37°C. for five days. Over this time the MBP was monitored for the loss ofAF488 by ELISA (FIG. 8 ). The sandwich ELISA procedure includedMBP-AF488 capture on an α-MBP-coated microtiter plate followed bydetection of covalently bound AF488 using an α-AF488 antibody and anHRP-conjugated secondary antibody. Over the course of five days, noappreciable hydrolysis of the azacarboline conjugate was observed. Incontrast, the oxime conjugate of AF488 decomposed within one day.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A conjugate comprising: at least one modified amino acid residue offormula (111):

wherein n is 0 or 1; R₁ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R₂and R₃ are each independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; X₁,X₂, X₃ and X₄ are each independently selected from C, N, O and S; Y₁,Y₂, Y₃, and Y₅ are each independently selected from hydrogen, halogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl,alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substitutedthioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl; L is an optional linker, wherein one of W₁ and W₂ is apolypeptide and the other is a drug or a detectable label.
 2. (canceled)3. (canceled)
 4. The conjugate of claim 1, wherein n is
 1. 5. Theconjugate of claim 1, wherein R₂ and R₃ are each independently selectedfrom alkyl and substituted alkyl.
 6. The conjugate of claim 1, whereinR₂ and R₃ are each methyl.
 7. The conjugate of claim 1, wherein X₁, X₂,X₃ and X₄ are each C.
 8. The conjugate of claim 1, wherein Y₁, Y₂, Y₃and Y₅ are each H.
 9. The conjugate of claim 1, wherein L is present andcomprises a group selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino,alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.
 10. The conjugate of claim1, wherein L is present and comprises a polymer.
 11. The conjugate ofclaim 10, wherein the polymer is a polyethylene glycol.
 12. Theconjugate of claim 1, wherein the detectable label comprises afluorophore.
 13. The conjugate of claim 1, wherein W₁ is the drug or thedetectable label, and W₂ is the polypeptide.
 14. The conjugate of claim1, wherein W₁ is the polypeptide, and W₂ is the drug or the detectablelabel.
 15. (canceled)
 16. (canceled)
 17. (canceled)
 18. The conjugate ofclaim 1, wherein the conjugate comprises at least one modified aminoacid residue of formula (IIIa):

19.-41. (canceled)
 42. A pharmaceutical composition comprising: aconjugate of claim 1; and a pharmaceutically acceptable excipient.
 43. Amethod of delivering a conjugate to a subject, the method comprising:administering to the subject an effective amount of a conjugate ofclaim
 1. 44. A method of treating a condition in a subject, the methodcomprising: administering to the subject having the condition atherapeutically effective amount of a pharmaceutical compositioncomprising a conjugate of claim 1, wherein the administering iseffective to treat the condition in the subject.